MOLECULAR AND CELLULAR BIOLOGY, May 2001, p. 2991–3000
0270-7306/01/$04.00⫹0 DOI: 10.1128/MCB.21.9.2991–3000.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Vol. 21, No. 9

Identification of Liver X Receptor-Retinoid X Receptor as an
Activator of the Sterol Regulatory Element-Binding
Protein 1c Gene Promoter
TOMOHIRO YOSHIKAWA,1 HITOSHI SHIMANO,2* MICHIYO AMEMIYA-KUDO,1 NAOYA YAHAGI,1
ALYSSA H. HASTY,1 TAKASHI MATSUZAKA,2 HIROAKI OKAZAKI,1 YOSHIAKI TAMURA,1
YOKO IIZUKA,1 KEN OHASHI,1 JUN-ICHI OSUGA,1 KENJI HARADA,1
TAKANARI GOTODA,1 SATOSHI KIMURA,1 SHUN ISHIBASHI,1
2
AND NOBUHIRO YAMADA
Department of Metabolic Diseases, University of Tokyo, Bunkyo-ku, Tokyo 113-8655,1 and Department ofInternal Medicine, Institute of Clinical Medicine, University of Tsukuba, Ibaraki 305-8575,2 Japan
Received 23 October 2000/Returned for modification 19 December 2000/Accepted 9 February 2001

In an attempt to identify transcription factors which activate sterol-regulatory element-binding protein 1c(SREBP-1c) transcription, we screened an expression cDNA library from adipose tissue of SREBP-1 knockout
mice using a reporter gene containing the 2.6-kb mouse SREBP-1 gene promoter. We cloned and identified theoxysterol receptors liver X receptor (LXR␣) and LXR␤ as strong activators of the mouse SREBP-1c promoter.
In the transfection studies, expression of either LXR␣ or -␤ activated the SREBP-1c promoter-luciferase genein a dose-dependent manner. Deletion and mutation studies, as well as gel mobility shift assays, located an
LXR response element complex consisting of two new LXR-binding motifs which showed high similarity to an
LXR response element recently found in the ABC1 gene promoter, a reverse cholesterol transporter. Additionof an LXR ligand, 22(R)-hydroxycholesterol, increased the promoter activity. Coexpression of retinoid X
receptor (RXR), a heterodimeric partner, and its ligand 9-cis-retinoic acid also synergistically activated the
SREBP-1c promoter. In HepG2 cells, SREBP-1c mRNA and precursor protein levels were induced by treatment
with 22(R)-hydroxycholesterol and 9-cis-retinoic acid, confirming that endogenous LXR-RXR activation can
induce endogenous SREBP-1c expression. The activation of SREBP-1c by LXR is associated with a slightincrease in nuclear SREBP-1c, resulting in activation of the gene for fatty acid synthase, one of its downstream
genes, as measured by the luciferase assay. These data demonstrate that LXR-RXR can modify the expressionof genes for lipogenic enzymes by regulating SREBP-1c expression, providing a novel link between fatty acidand cholesterol metabolism.
of target genes involved in both cholesterol and fatty acidsynthesis (30, 31).
Lipogenic enzymes, which are involved in energy storage
through synthesis of fatty acids and triglycerides, are coordinately regulated at the transcriptional level during different
metabolic states (9, 11). Recent in vivo studies demonstrated
that SREBP-1c plays a crucial role in the dietary regulation ofmost hepatic lipogenic genes, whereas SREBP-2 is actively
involved in the transcription of cholesterogenic enzymes (13).
These include studies of the effects of the absence or overexpression of SREBP-1 on hepatic lipogenic gene expression (30,31, 33), as well as physiological changes of SREBP-1c protein
in normal mice after dietary manipulation such as placementon high-carbohydrate diets, polyunsaturated fatty acid-enriched diets, and fasting-refeeding regimens (12, 17, 37, 40, 41).
The similar coordinated changes in SREBP-1c and lipogenic
gene expression upon fasting and refeeding were also observedin adipose tissue (18). In fat tissue, SREBP-1c (ADD1) appears to be involved in adipocyte differentiation and insulin
resistance (19, 35). Recent studies suggest that insulin or insulin-facilitated glucose uptake mediates lipogenesis through
SREBP-1c induction (7, 8, 10, 21, 34). Previous reports on theregulation of SREBP-1c have all demonstrated the induction
to be at the mRNA level. Up-regulation of hepatic SREBP-1
mRNA was observed in the livers of rodents on a fastingrefeeding regimen or a chronic high-carbohydrate diet and in

Sterol-regulatory element (SRE)-binding proteins (SREBPs)
are transcription factors which belong to the basic helix-loophelix leucine zipper family (3–5). In contrast to other membersof this family, SREBPs are synthesized as precursor proteins
which remain bound to the endoplasmic reticulum and thenuclear envelope in the presence of sufficient sterol concentrations. Upon sterol deprivation, the precursor protein undergoes a sequential two-step cleavage process to release the NH2terminal portion (28). This mature SREBP then enters thenucleus and activates the transcription of genes involved incholesterol and fatty acid synthesis by binding to SREs or topalindromic sequences called E boxes within their promoter
regions (20, 39). Three forms of SREBP have been characterized: SREBP-1a and -1c (also known as ADD1) (14, 38, 43)
and SREBP-2. It has been shown that all of the cultured cells
analyzed to date express primarily SREBP-2 and the SREBP1a isoform, whereas most organs, including the liver and adipose tissue, express predominantly SREBP-2 and the SREBP1c isoform (36). SREBP-1a is a stronger activator than SREBP-1c
due to its longer transactivation domain and has a wider range* Corresponding author. Mailing address: Department of Internal
Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1
Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Phone and fax: 81-29863-2170. E-mail: shimano-tky@umin.ac.jp.
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primary hepatocytes with a high-glucose medium (8, 10, 12,41). In contrast, down-regulation of SREBP-1c was observed inlivers from fasted rodents, from insulin-depleted diabetic rats
with streptozotocin treatment, and from mice on a diet containing polyunsaturated fatty acids (12, 17, 34, 37, 40, 41).
In contrast to SREBP-2, which mediates sterol regulation completely at the cleavage level through interaction with SREBP
cleavage-activating protein (SCAP) and site 1 protease, SREBP1c controls the transcriptional regulation of lipogenic enzymes
by self-regulating the nuclear concentration of its mature form,
which is highly correlated to its precursor and mRNA levels.
The initial analysis of the mouse SREBP-1c promoter identified two functional regions (1). One region is the SRE complex, which confers a response to SREBPs. Upstream of thisregion is an oxysterol-inducible region, the details of which
remain unknown. To investigate the relationship between the
promoter sequence and physiological regulation of SREBP-1c,
we sought to identify transcription factors that might regulate
the SREBP-1c promoter. In the screening of the SREBP-1cgene activators, we cloned and identified liver X receptors(LXR␣ [NR1H3] and LXR␤ [NR1H2]) as strong activators of
SREBP-1c gene promoter.
LXRs belong to a subclass of nuclear hormone receptors
that form obligate heterodimers with retinoid X receptors(RXRs) and are bound and activated by oxysterols (16, 22, 24,
27). LXRs regulate the expression of cholesterol 7␣-hydroxylase, the rate-limiting enzyme of the classic bile acid biosynthetic pathway (22, 25, 27); cholesterol ester transfer protein,which translocates cholesterol ester between lipoprotein fractions (23); and ATP-binding cassette transporter 1 (ABC1),
which modulates the reverse cholesterol transport from peripheral tissues and cholesterol absorption in the intestine (26).
Thus, LXR-RXR heterodimers serve as key regulators of cholesterol homeostasis. Identification of LXR-RXR as an activator of the SREBP-1c promoter reveals a new aspect of cholesterol and fatty acid metabolism.
MATERIALS AND METHODS
Materials and general methods. We obtained cholesterol, 22(R)-hydroxycholesterol [22(R)-HC], 25-hydroxycholesterol (25-HC), and 9-cis-retinoic acid
(9CRA) from Sigma; Redivue [␣-32P]dCTP (6,000 Ci/mmol) from Amersham
Pharmacia; restriction enzymes from New England BioLabs; and polyclonal
antibodies against mouse LXR␣ and -␤ (C19, sc-6063x) and RXR␣ (D-20,
sc-553x) from Santa Cruz Biotechnology, Inc.
Standard molecular biology techniques were used. DNA sequencing was performed with the PRISM dye terminator cycle sequencing kit and an ABI PRISM
310 genetic analyzer (Applied Biosystems).
Plasmids. Luciferase gene constructs containing 2.6-kb and 550-bp fragmentsof the mouse SREBP-1c promoter (pBP1c2600-Luc and pBP1c550-Luc, respectively) were prepared as described previously (1). Human RXR␣ expression
plasmid pRXR was a kind gift from D. J. Mangelsdorf. Other SREBP-1 promoter-luciferase constructs were produced by PCR using pBP1c550-Luc as a
DNA template and subcloning the PCR products into the pGL2 basic or pGL2
promoter vector (Promega). The PCR primers used were as follows: 5⬘ primers
BP1c408-Luc (5⬘-GGCCAGGAGTGGGTAAA-3⬘), BP1c357-Luc (5⬘-GTGTGCGAACGAACCAG-3⬘), BP1c296-Luc (5⬘-GGATTCCGGACCCAGGCT-3⬘),
BP1c249-Luc (5⬘-GGGTTGGGACGACAGTGA-3⬘), BP1c197-Luc (5⬘-GGC
GCAGACGCGGTTA-3⬘), BP1c148-Luc (5⬘-GGGAGAAACCCGAGCT-3⬘),
BP1c186-Luc (5⬘-GGACGCCCGCTAGTAACC-3⬘), BP1cLXREa-Luc (5⬘-GA
CAGTGACCGCCAGTAACCC-3⬘), and BP1cLXREaM-Luc (5⬘-GACAGAGT
CCGCCAGAATCCC-3⬘) and 3⬘ primers BP1c41-Luc (5⬘-TAAGAGCTCGGT
ACCTCCCCTAGGGC-3⬘), BP1c149-Luc (5⬘-GGTGCTCTGAATGGGGC-3⬘),
BP1cLXREb-Luc (5⬘-CCGGGGTTACTAGCGGGC-3⬘), and BP1cLXREbM-

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Luc (5⬘- CCGGGGATTCTAGCGGGC-3⬘). The primers were tailed with an
SmaI site (5⬘ primers) and a KpnI site (3⬘ primers).
Preparation of expression library and screening of transactivator of the
SREBP-1c promoter. For preparation of the cytomegalovirus (CMV) promoterexpression vector, the SmaI site of CMV7 (2) was converted to a NotI site usinga NotI linker and designated CMV7-NotI. Total RNA was extracted from adipose tissues from three SREBP-1 knockout mice (33) using Trizol (Life Technologies, Inc.). Poly(A)⫹ RNA was prepared from total RNA by two cycles ofchromatography on oligo(dT) (mRNA Purification Kit; Amersham Pharmacia
Biotech). A size-fractionated, directional cDNA library with SalI and NotI cohesive ends at the 5⬘ and 3⬘ termini, respectively, was constructed from 5 ␮g ofpoly(A)⫹ RNA using a Superscript Plasmid Kit (Life Technologies, Inc.). Sizefractionated cDNA (⬎1.0 kb) was ligated with expression vector pCMV7-NotI
using the protocol and reagents supplied with the Superscript kit. The ligation
reaction of cDNAs and the vectors digested with SalI and NotI was transformed
into electrocompetent DH5␣ cells and aliquoted into subpools containing approximately 1,500 clones. The transcriptional activity of each pool (1.5 to 2.0 ␮gof total DNA) for the SREBP-1c promoter was assayed by transfection into
human embryonic kidney (HEK) 293 cells with pBP1c2600-Luc (0.25 ␮g) andcontrol ␤-galactosidase reference plasmid pCMV-␤gal (0.1 ␮g; Promega) as
described below.
Transfections and luciferase assays. HEK 293 and HepG2 cells were grown at
37°C in an atmosphere of 5% CO2 in Dulbecco’s modified Eagle’s medium(DMEM) containing 25 mM glucose, 100 U of penicillin per ml, and 100 ␮g ofstreptomycin sulfate per ml supplemented with 10% fetal calf serum (FCS).
Transfection studies were carried out with cells plated on 12-well plates as
described previously (1). The indicated amount of each expression plasmid wastransfected simultaneously with a luciferase reporter plasmid (0.25 ␮g) andpCMV-␤gal or pSV-␤gal (0.1 to 0.4 ␮g). The total amount of DNA in eachtransfection was adjusted to 1.5 ␮g/well with pCMV7-NotI vector DNA. Ligands
for LXR and RXR were dissolved in ethanol and added to cells after transfectionin DMEM with 10% FCS. For suppressed conditions, DMEM with 10% FCS,
cholesterol (10 ␮g/ml), and 25-HC (1 ␮g/ml) was used to suppress endogenous
SREBP activity. The amount of luciferase activity in transfectants was measured
and normalized to the amount of ␤-galactosidase activity as measured by standard kits (Promega).
Gel mobility shift assays. Gel mobility shift assays were done as previously
described (1). Briefly, nuclear extracts were prepared from HEK 293 cells as
described previously (31). Double-stranded oligonucleotides used in gel mobility
shift assays were prepared by annealing both strands of each LXR response
element (LXRE) of the LXRE complex in the SREBP-1c promoter (LXREa[5⬘-CAGTGACCGCCAGTAACCCCAGC-3⬘] and LXREb [5⬘-GGACGCCCG
CTAGTAACCCCGGC-3⬘]), labeled with [␣-32P]dCTP by Klenow enzyme, andpurified on Sephadex G50 columns. The labeled probes (3,000 to 10,000 cpm)
were incubated with nuclear extracts (3 ␮g) in a mixture (20 ␮l) containing 10
mM Tris-HCl (pH 7.6), 50 mM KCl, 0.05 mM EDTA, 2.5 mM MgCl2, 8.5%
glycerol, 1 mM dithiothreitol, poly(dI-dC) at 0.5 ␮g/ml, 0.1% Triton X-100, andnonfat milk at 1 mg/ml for 30 min on ice. The DNA-protein complexes wereresolved on a 4.6% polyacrylamide gel at 140 V for 1 h at 4°C. Gels were dried
and exposed to a BAS2000 filter with BAStation software (Fuji Photo Film Co.,
Ltd.). For competition experiments, at least a 100-fold molar excess of unlabeled
DNA relative to labeled DNA was added to the reaction mixture before additionof the labeled probe. In the supershift experiments, the gel shift reactions werefirst incubated for 1 h with 2 to 10 ␮g of polyclonal antibodies against LXR␣ and-␤ or RXR␣ on ice.
Nuclear extract preparation and immunoblot analysis. Nuclear extracts from
HepG2 cells were prepared as described previously (33). The samples of 30 ␮gof nuclear protein were subjected to immunoblot analysis with mouse immunoglobulin G (IgG) against human SREBP1 (30) or rabbit IgG against human
SREBP1c and SREBP2 (32, 33), followed by horseradish peroxidase-linked
mouse or rabbit IgG and the ECL kit (Amersham Pharmacia Biotech).
Total RNA preparation and blot hybridization with cDNA probes. Total RNA
was extracted from mouse livers, adipose tissues, and HepG2 cells using
TRIZOL Reagent (Life Technologies, Inc.). Equal aliquots of total RNA frommice in each group were pooled (total, 10 ␮g), subjected to formalin-denatured
agarose electrophoresis, and transferred to nylon membrane (Hybond N; Amersham Pharmacia Biotech). Blot hybridization was performed with the cDNA
probes labeled with [␣-32P]CTP (6,000 Ci/mmol) using the Megaprime DNA
Labeling System (Amersham Pharmacia Biotech). The cDNA probes for mouse
LXRs were prepared by digesting the cloned full-length LXR cDNAs (CMVLXR␣ and -␤) with SalI and with EcoRI and NotI, respectively. The cDNA
probes for human SREBP1, human SREBP2, human fatty acid synthase (FAS),
and human acidic ribosomal phosphoprotein P0 (36B4) were prepared as pre-VOL. 21, 2001

ACTIVATION OF SREBP-1c GENE PROMOTER BY LXR-RXR

FIG. 1. Screening of an SREBP-1 knockout mouse adipose tissue expression library to identify cDNA clones which activate the
SREBP-1c promoter. An expression cDNA library from the adipose
tissues of SREBP-1-null mice was constructed as described in Materials and Methods. A luciferase reporter gene containing the mouse
SREBP-1c promoter (2.6 kb), pBP1c2600-Luc, and each subpool containing approximately 1,500 cDNA clones were cotransfected into
HEK 293 cells with pSV-␤gal as a reference plasmid. Luciferase activity was measured and normalized by ␤-galactosidase activity. Afterseveral rounds of screening, six positive single clones were isolated
from the library (three LXR␣ and three LXR␤). A representative
result from the initial screening is shown.

viously described (33). The resulting bands were visualized by exposure to
BAS2000 filters with BAStation software (Fuji Photo Film Co., Ltd.).

RESULTS
Our previous promoter analysis of the SREBP-1c gene 5⬘
flanking region identified an SRE complex which confers an autoloop response to nuclear SREBPs and another upstream region
which was induced by the presence of oxysterols (1; see Fig. 4).
The presence of two regions with opposing sterol regulation
makes SREBP-1c gene expression very complex. The initial goalof this study was to isolate transcription factors that activate
SREBP-1c expression. We used an expression cloning strategy ofplacing the luciferase gene containing 2.6 kb of the 5⬘ flanking
region of the mouse SREBP-1c promoter in reporter gene assays.
We previously observed that adipose tissues from SREBP-1
knockout mice highly expressed the aberrant SREBP-1c mRNA
from the intact promoter of the disrupted SREBP-1c gene (33).
Therefore, we assumed that these tissues might also abundantly
express the transcription factors we were seeking. From mRNAsof these tissues, we constructed an expression cDNA library under the CMV promoter. Screening was performed using luciferase assays after the cotransfection of subpools of the library andthe SREBP-1c promoter luciferase reporter gene into 293 cells.
Figure 1 shows a representative result of the initial screening of
subpools of the library. We screened roughly 210,000 clones andpicked up six positive subpools of 1,500 clones that exhibited twoto threefold increases in luciferase activity of the SREBP-1c promoter reporter compared to background levels. After several
rounds of screening, six single clones were isolated from these
subpools. Each of these isolated clones increased the luciferase
activity 10- to 20-fold above the background. DNA sequencing
revealed that three of these six clones were LXR␣ and the remaining three were LXR␤. No other clones were identified in thissystem. Both LXR␣ and -␤ clones contained full-length cDNAs,

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FIG. 2. Northern blot analysis of LXRs from livers and white adipose tissues (WAT) from mice fasted and refed. Total RNA wasprepared and pooled (10 ␮g) equally from either livers or adipose
tissues of normal mice fasted (F) or refed (R) after fasting as described
previously (41), blotted to a nylon membrane, and hybridized with theindicated cDNA probes. A cDNA probe for 36B4 (acidic ribosomal
phosphoprotein P0) was used as a loading control.

were assumed to express their complete respective proteins in theassays, and were designated pCMV-LXR␣ and -␤, respectively.
From a standpoint of nutritional regulation of these genes, we
examined the expression of LXR␣ and -␤ in lipogenic organs, alocation where the expression of SREBP-1c is suppressed by
fasting and highly upregulated by refeeding. As shown in Fig. 2,
both LXR␣ and -␤ were highly expressed in both livers andadipose tissues; however, the mRNA levels were not markedly
changed by these dietary manipulations. Thus, expressions of
LXRs in the lipogenic organs were not subjected to the nutritional change at fasting and refeeding, unlike SREBP-1c andperoxisome proliferator-activated receptor ␣.
Studies were next focused on the effects of LXR␣ and -␤ on the
SREBP-1c promoter. As an initial step, we tested the dose-dependent effects of LXR␣ and -␤ on the transcriptional activity of
the SREBP-1c promoter (Fig. 3, left panel). Expression of both
LXR␣ and -␤ resulted in a dose-dependent increase in the luciferase activity of the 2.6-kb SREBP-1c promoter in the range of
0.03 to 1 ␮g of DNA. SREBP-1c promoter activity was also
induced by addition of 22(R)-HC, a well-established ligand for
LXRs, to the medium in transfection studies with HEK 293 cells(Fig. 3, right panel). A marked dose-dependent increase in theluciferase activities was observed at 22(R)-HC concentrations between 1 and 10 ␮M, a dose range commonly used for activationof LXR. These data suggest that either the endogenous LXR␣ or-␤, or both, in the transfected cells was activated by 22(R)-HC and
was able to induce SREBP-1c promoter activity. 25-HC was asignificant, but much weaker, activator, whereas cholesterol wasessentially inactive. To evaluate the effect of free cholesterol,different doses of cholesterol were added to cells cultured indelipidated serum and the luciferase activity was measured. Up to
10 ␮M cholesterol did not activate the SREBP-1c promoter;
however, 30 ␮M cholesterol slightly increased the luciferase activity (data not shown). These results are consistent with the2994

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FIG. 3. Dose-dependent activation of SREBP-1c promoter by LXRs
(left panel) and their ligands (right panel). (Left panel) pBP1c2600Luc was cotransfected into HEK 293 cells with a positive clone expressing LXR from the screening (pCMV-LXR␣ or pCMV-LXR␤),an empty vector (CMV-7) used as a control, or pSV-␤gal, which wasused as a reference plasmid. (Right panel) Ligands for LXR or ethanol(used as a control) were added to the cells after transfection of
pBP1c2600-Luc and pSV-␤gal in medium with 10% fetal bovine serum
24 h prior to the assay. After the incubation, luciferase activity wasmeasured and normalized to ␤-galactosidase activity. The fold induction of luciferase activity by LXRs or their ligands (means ⫾ standard
deviations in the left panel [n ⫽ 3]; means in the right panel [n ⫽ 2])compared to that of controls is shown. Cho, cholesterol.

previously reported affinities of these oxysterols for LXRs (15, 16,
22).
To identify the element in the SREBP-1c promoter that was
responsible for LXR activation, we compared the LXR activationof the luciferase constructs with different sizes of the SREBP-1c
promoter. Our previous study identified the sterol-inducible region between bp ⫺550 and ⫺90 (1; Fig. 4). Since LXR has beenshown to be activated by sterols (15, 16, 22), 25-HC, and choles-

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terol, compounds commonly used for a sterol-suppressive condition, it was reasonable to assume that this region could be an
LXR activation site. Initial luciferase assays with BP1c2600-Luc,
BP1c550-Luc, and BP1c90-Luc demonstrated that LXR activation was observed with BP1c2600 and BP1c550, but not with
BP1c90, confirming the presence of an LXR activation site between bp ⫺550 and ⫺90 (Fig. 4). Figure 5 shows luciferase activities after LXR␣ or -␤ coexpression in sequential-deletioncontaining SREBP-1c promoter constructs assembled to identify
the LXR activation sites. In the sterol-inducible region, we founda sequence at bp ⫺377 to ⫺362 which was similar to the LXRE(GGTCCAGGCAAGTTCT) (1). However, as shown in Fig. 5B,
deletion of this sequence did not affect the LXR activation of the
SREBP-1c promoter, indicating that this sequence was not involved. In contrast, considerable reduction in both LXR␣ and -␤
activation was observed when two regions, bp ⫺249 to ⫺187 andbp ⫺186 to ⫺148 were deleted. Loss of both regions essentiallyabolished the LXR activation, suggesting that these regions are
crucial. As depicted in Fig. 5A, each candidate region contains a
DR-4 sequence which was similar to the inverted LXRE. While
this report was being prepared; a detailed promoter analysis of
the ABC1 gene containing a new LXR-binding sequence in an
inverted orientation was reported (6; Fig. 5A). LXREa, an upstream element of the two LXREs in the SREBP-1c promoter
closely matched this new sequence (9 out of 10 bases in the two
direct repeats), and the other (LXREb) also showed significant
similarity (7 out of 10 bases).
To show direct binding of LXRs to LXREa or LXREb,
gel mobility shift assays were performed (Fig. 6). A labeled
LXREa or LXREb probe was shifted by incubation of nuclear
extracts from cells in which LXR␣ or -␤ and RXR were overexpressed. Coexpression of RXR was required for the binding

FIG. 4. Activation of SREBP-1c promoter by LXR via the oxysterol-inducible region in the promoter. The previously identified oxysterolinducible region and the SRE complex in the SREBP-1c promoter are illustrated at the top. pBP-1c2600-Luc and two shorter versions, pBP1c550and pBP1c90-Luc, were prepared and used for transfection into 293 cells as described in Materials and Methods. Luciferase activity was measured
and normalized to ␤-galactosidase activity. Fold induction of luciferase activity (mean ⫾ standard deviation) by LXRs is shown.

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FIG. 5. Identification of LXREs by deletional and mutational analysis of the oxysterol-inducible region in the SREBP1c-promoter. (A) DNA
sequencing of the oxysterol-inducible region of the SREBP1c promoter identified two elements, which were designated mLXREa and -b. Theseelements are highly similar to the LXRE of the human ABC1 promoter. (B) Sequential deletion and mutation analysis of the oxysterol-inducible
region was performed with reporter genes with the indicated sizes and positions of the promoter. HEK 293 cells were transfected with each reporter
plasmid, pCMV-LXR, and reference plasmid pSV-␤gal and thereafter cultured for 24 h in medium with 10% fetal bovine serum. Luciferase activity
was measured and normalized to ␤-galactosidase activity. Fold induction of luciferase activity by LXRs (means ⫾ standard deviations) is shown.
SV40, simian virus 40.

of LXRs to LXREs (data not shown). Preincubation of the reaction mixture with antibodies against LXR␣ and -␤ or RXR␣caused a supershift of the LXR-RXR DNA complex, confirming the specific binding of LXR-RXR to these LXREs, although the shifts caused by these antibodies were partial, as previously described (23). Competition assays suggest that LXREb
has a slightly higher affinity for LXRs than does LXREa.
Based upon these data, we tentatively designated the region
between bp ⫺249 and bp ⫺149 containing the two LXR-bindingsites an LXRE complex (Fig. 6B). An LXRE complex enhancer
construct (pLXRE-Luc) in which the LXRE complex was inserted upstream of simian virus 40 promoter luciferase gene
(pGL2 promoter vector) was prepared and tested for activation
by LXR. Dose dependency and the effect of 22(R)-HC were
essentially the same as those observed in the 2.6-kb SREBP-1c

promoter construct (Fig. 7), indicating that this region basically
represents the whole response of the SREBP-1c promoter to
LXR. Mutation analysis was also performed on these two LXREs
using the LXRE complex enhancer construct (pLXRE-Luc) (Fig.
5B). Mutation of either LXREa or LXREb significantly reduced
the induction of luciferase activity by LXR expression. Mutationof both LXREs essentially abolished the effect of LXR. These
results confirmed the role of both LXREs in LXR activation of
the SREBP-1c promoter, although LXREb seemed relatively
more important.
Since LXRs are obligated to heterodimerize with RXR in order to activate their target genes, the effects of coexpression of
RXR and its ligand on LXR activation of LXRE-Luc were investigated (Fig. 8). Addition of 22(R)-HC or 9CRA to the medium caused some increase in this enhancer activity, and both

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FIG. 6. Binding of LXR to LXREs in the SREBP1c promoter as measured by gel mobility shift assay. (A) LXREa and LXREb in the SREBP1c promoter were labeled and incubated with nuclear extracts from transfected 293 cells in which LXR␣ or -␤ and RXR were overexpressed. A
molar excess of unlabeled LXREa or -b fragment and antibody against LXR␣ and -␤ or RXR␣ were added to the incubations in competition andsupershift assays, respectively. The DNA-protein complexes were resolved in a 4.8% polyacrylamide gel. The shifted and supershifted LXRE
probes are indicated by thick and thin arrows, respectively. NR, nuclear extract from mock-transfected cells used as a negative control. (B) LXREa
and -b in the oxysterol-inducible region of the SREBP-1c promoter are shown.

ligands caused synergistic induction, presumably due to endogenous expression of LXR and RXR in 293 cells (Fig. 8, no receptor). RXR expression alone showed only slight additional effects
on the activities of the respective ligands. LXR␣ or -␤ expression
alone caused an ⬃15-fold increase in the enhancer activity,
which was consistent with the original result obtained with
BP1c2600Luc. LXR␣ (or -␤) expression with 22(R)-HC synergistically induced activation of the promoter 60-fold (50fold), with further induction by RXR expression and 9CRA up
to 170-fold (155-fold). There was no significant difference intranscriptional activity between LXR␣ and LXR␤. Finally,
LXR␣ (or -␤)-RXR coexpression caused a further additional
effect, with a maximal effect of 370-fold (300-fold) induction in
the presence of both 22(R)-HC and 9CRA. These data demonstrate that LXR and RXR and their ligands activate the
SREBP-1c promoter activity in a synergistic fashion throughthe LXRE enhancer complex.
To evaluate the consequences of LXR-RXR activation, theeffects of LXR and/or RXR ligands on endogenous SREBP-1c
expression were estimated in HepG2 cells. As shown in Fig. 9,

SREBP-1c precursor protein, as estimated by immunoblot analysis of cell membranes with mouse SREBP-1c-specific antibody,was barely detectable in HepG2 cells but was slightly induced byaddition of the LXR ligand 22(R)-HC. Interestingly, the RXR
ligand 9CRA induced the SREBP-1c protein to a greater extent
than did 22(R)-HC and combination of the two ligands had an
additive effect. LXR-RXR induction of SREBP-1, as demonstrated by an SREBP-1 antibody that detects both SREBP-1a and-1c, was less marked, suggesting that SREBP-1a was not regulatedby those ligands. SREBP-2 precursor protein was not regulated by
LXR either. The data suggest that LXR-RXR activation is specific to SREBP-1c. Nuclear SREBP-1c protein was detected innuclear extracts from the HepG2 cells treated with both
22(R)-HC and 9CRA, demonstrating that induced SREBP-1c
expression by activation of LXR-RXR could result in the cleavage and movement of the mature form of SREBP-1c protein to
the nucleus.
Northern blot analysis demonstrated that addition of both
22(R)-HC and 9CRA induced mRNAs of the SREBP-1 geneand the gene for FAS, a representative SREBP-1 target lipo-

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ACTIVATION OF SREBP-1c GENE PROMOTER BY LXR-RXR

FIG. 7. Dose-dependent activation of the LXRE complex enhancer in
the SREBP-1c promoter by LXRs (left panel) and their ligands (rightpanel). The LXRE complex (LXREa and -b, bp ⫺249 to ⫺148) in the
SREBP-1c promoter was fused to a luciferase reporter plasmid whichcontained a simian virus 40 promoter (pGL2 promoter vector; Fig. 5) toestimate LXRE enhancer activity (pLXRE-Luc). (Left panel) pLXRELuc was cotransfected into HEK 293 cells with pCMV-LXR␣, pCMVLXR␤, or an empty vector (CMV-7, used as a control) and pSV-␤gal,
which was used as a reference plasmid. (Right panel) Ligands for LXR orethanol (used as a control) was added to the cells after transfection ofpLXRE-Luc and pSV-␤gal 24 h prior to the assay. After the incubation,luciferase activity was measured and normalized to ␤-galactosidase activity. Fold induction of luciferase activity by LXRs or their ligands(means ⫾ standard deviations in the left panel [n ⫽ 3]; means in the rightpanel [n ⫽ 2]) compared to that of the control is shown. Cho, cholesterol.

genic gene, but not SREBP-2 gene mRNA (Fig. 10). HepG2
cells were transfected with the FAS promoter-luciferase geneto evaluate the transcriptional activity of LXR-induced nuclear
SREBP-1c. As shown in Fig. 11, addition of 22(R)-HC and
9CRA to the medium caused only a marginal increase in the
FAS-Luc activity. However, coexpression of LXR␣ and -␤ withthese ligands markedly increased the luciferase activity up to
one-third of the level of activation caused by by direct overexpression of nuclear SREBP-1c (CMV-SREBP1c). This induction was significant even in the presence of 25-HC and cho-

2997

FIG. 9. Induction of SREBP-1c protein by LXR-RXR ligands in
HepG2 cells as measured by immunoblot analysis. HepG2 cells wereincubated with the indicated ligands for LXR or RXR for 24 h. In
some groups, 1 ␮g of 25-HC per ml and 10 ␮g of cholesterol per mlwere also added to the medium to suppress sterol-regulatory cleavage
activity of SREBPs (sterol-suppressed condition). After the incubation, nuclear extracts and membranes were prepared from the cells andaliquots (30 ␮g) of protein were subjected to immunoblot analysis
using antibodies against SREBP-1c, SREBP-1a and -1c, and SREBP-2.
The bands were visualized with the ECL system. The values below thebands refer to relative fold changes in protein levels. Lanes: 1, ethanol;
2, 22(R)-HC; 3, 9CRA; 4, 22(R)-HC plus 9CRA; 5, ethanol in a sterolsuppressed condition; 6, 22(R)-HC plus 9CRA in a sterol-suppressed
condition.

lesterol, a condition that suppresses endogenous SREBP-1aand -2 activities by inhibiting the sterol-regulatory cleavage of
SREBPs. These data demonstrate that LXR-RXR inductionof SREBP-1c can potentially increase its target gene expression regardless of sterol regulation.
DISCUSSION

FIG. 8. Synergistic activation of the LXRE complex in the SREBP1c promoter by LXRs, RXR, and their ligands. The luciferase reporter
construct containing the LXRE complex in the mouse SREBP-1c
promoter (pLXRE-Luc) was cotransfected into HEK 293 cells with
pCMV-LXR␣ or -␤, pRXR, or both and pSV-␤gal. After the transfection, the cells were incubated in the absence or presence of the ligand 22(R)-HC, 9CRA, or both for 24 h. Luciferase activity wasmeasured and normalized to ␤-galactosidase activity. Fold induction(means ⫾ standard deviations) compared to that of the control (anempty expression vector with no addition of ligands) is shown.

The current studies clearly demonstrate that LXR-RXR activates SREBP-1c expression by binding to newly identified
LXR-binding sites in the SREBP-1c promoter. In our expression cloning system from mouse adipose tissue with a reporter
gene containing the native SREBP-1c promoter (2.6 kb), we
could isolate only LXR␣ and -␤, suggesting that LXRs areabundant and potent activators of the SREBP-1c promoter.
The LXRE complex, composed of two inverted DR4-type
LXREs at approximately a 30-bp interval. Both binding sites
contributed to activation of the SREBP-1c promoter. The promoters of the LXR target genes which have been analyzed todate contain only a single LXRE (6, 22, 23). The presence of
two copies of LXREs in the LXRE complex could make theresponse of the SREBP-1c gene to LXR-RXR strong.
The importance of LXR for expression of SREBP-1 hasalready been recognized in a previous report on LXR␣ knockout mice (25). In the LXR␣ knockout mice, induction of cho-

2998

YOSHIKAWA ET AL.

FIG. 10. SREBP-1, -2, and FAS mRNA levels in HepG2 cells
treated with LXR or RXR ligands as measured by Northern blot
analysis. HepG2 cells were incubated with the indicated ligands for
LXR or RXR for 24 h. In some groups, 25-HC at 1 ␮g/ml and cholesterol at 10 ␮g/ml were added to the medium to suppress the sterolregulatory cleavage activity of SREBPs (sterol-suppressed condition).
Total RNA was prepared and pooled (10 ␮g) equally from HepG2
cells, blotted to a nylon membrane, and then hybridized with theindicated cDNA probes. The values below the bands refer to relative
fold changes in mRNA levels normalized relative to the 36B4 signal.
Lanes: 1, ethanol; 2, 22(R)-HC; 3, 9CRA; 4, 22(R)-HC plus 9CRA;
5, ethanol in a sterol-suppressed condition; 6, 22(R)-HC plus 9CRA ina sterol-suppressed condition. A cDNA probe for 36B4 (acidic ribosomal phosphoprotein P0) was used as a loading control.

lesterol 7␣-hydroxylase was impaired, resulting in hepatic
accumulation of cholesterol when dietary cholesterol was consumed. One intriguing observation in the livers of these mice
was a considerable reduction of SREBP-1 mRNA and protein
levels, leading to a decrease in some lipogenic enzyme mRNAlevels. Our current data on activation of SREBP-1c expression by LXRs can explain this observation. Furthermore, the
LXR␣-deficient mouse data suggest that LXR␣ is an important regulator of SREBP-1c expression in the liver because
LXR␤ cannot entirely compensate for the absence of LXR␣. It
would be interesting to see a tissue survey of SREBP-1c expression in LXR␣- and -␤-deficient mice, as well as mice doubly deficient in both LXRs, to estimate the contribution ofthese two factors to SREBP-1c expression in different tissues.
It is intriguing to speculate about the physiological significanceof SREBP-1c induction by LXR. Recent studies identifying thegenes for 7␣-cholesterol hydroxylase (25), cholesterol ester transfer protein (23), and ABC1 (6, 26) as target genes of LXRs
suggest that LXR-RXR seems to play an important role in cellular adaptation to the accumulation of excess cholesterol byenhancing catabolism of cholesterol into bile acid (23, 25) or bypromoting cholesterol efflux (26, 29). SREBP-1c is a transcription
factor for lipogenesis, and its induction should cause an increasein the synthesis of fatty acids. These fatty acids can then provide
a source for acyl residues to produce cholesterol esters fromcholesterol and protect cells from the cytotoxic effect of freecholesterol accumulation (Fig. 12A). On the other hand, in cellswith a cholesterol overload leading to activation of the cholesterol
efflux system, cholesterol synthesis is negatively regulated through

MOL. CELL. BIOL.

FIG. 11. Activation of the FAS promoter by LXRs and their ligands. A luciferase reporter construct containing the rat FAS promoter (31) and reference plasmid pSV-␤gal with empty plasmid
pCMV7 or expression plasmids pCMV-LXR and pRXR were transfected into HepG2 cells. After the transfection, ligands for LXR andRXR or ethanol (used as a negative control) was added to the cells,followed by a 24-h incubation in the medium with 10% fetal bovine
serum. In some sets, 25-HC at 1 ␮g/ml and cholesterol at 10 ␮g/ml
were also added to the medium to suppress the sterol-regulatory cleavage activity of SREBPs (sterol-suppressed condition). Luciferase activity was measured and normalized to ␤-galactosidase activity. Fold
induction (means ⫾ standard deviations) by LXRs is shown. pCMVnSREBP-1c, an expression plasmid of human nuclear SREBP-1c wasused as a positive control for FAS promoter activation (BP-1c). L␣⫹R,
LXR␣ plus RXR; 22⫹9, 22(R)-HC plus 9CRA.

suppression of cleavage of SREBPs by a sterol-sensing and regulation system consisting of SCAP and site 1 protease. This
reduction in SREBP cleavage activity should counterbalance
the effect of LXR induction of the SREBP-1c precursor protein. However, our recent findings suggest that cleavage of
SREBP-1c is not as tightly regulated by sterols as is that of
SREBP-2 in liver cell lines, suggesting that nuclear SREBP-1c
has a chance to liberate its nuclear active form into the nucleus
even in a state of cholesterol accumulation (10). This is supported by our current finding that transfection of LXR-RXR
into HepG2 cells with addition of 22(R)-HC increased theamount of endogenous nuclear SREBP-1c and the resultant
luciferase activity of its downstream gene even in a sterolsuppressed condition (Fig. 11). Taken together, these data
allow us to hypothesize that LXR-RXR activation of SREBP1c might have a physiologic function situations of excess cellular cholesterol accumulation (Fig. 12A). While this would
result in an overall decrease in the cleavage of SREBPs, thelack of complete control of sterols over the SREBP-1c precursor, combined with the ability of LXRs to simulate SREBP-1c
expression, would ensure that a certain amount of nuclear
SREBP-1c was available to stimulate the production of fatty
acids, presumably to protect cells from the deposition of freecholesterol to a toxic level. On the other hand, when cells are
depleted of cholesterol, activation of the cleavage system will
increase nuclear SREBPs, including SREBP-1c, to maintainlipogenesis even in the absence of LXR activation (Fig. 12B).
As illustrated in Fig. 12, this multiple-complex system for
SREBP-1c promoter activity links SREBP-1c expression, andthus lipogenesis, partially to sterol regulation but also to someVOL. 21, 2001

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2999

FIG. 12. Dual regulation of SREBP-1c promoter by LXR-RXR and SREBPs. In the presence of excess amount of sterols, presumably a
concomitant increase in oxysterol levels activates the SREBP-1c promoter through the LXRE complex while the cleavage activity of SREBPs issuppressed (A). When cells are deprived of sterols, increased nuclear SREBPs activate the SREBP-1c promoter through the SRE complex (B).
The dual activation of the SREBP-1c promoter through LXRE and SRE complexes ensures the maintenance of lipogenesis irrespective of cellular
sterol levels.

levels of control irrespective of sterols. Considering that the listof SREBP-1 target genes seems to be expanding, it is also
possible that LXR activation of SREBP-1c could activate unknown target genes to cause as yet unidentified physiologicalconsequences.
Oxysterol receptors are involved in cholesterol catabolism (activation of 7␣-cholesterol hydroxylase for bile acid synthesis andABC1 for the reverse cholesterol transporter [6, 25, 26]), with theligands 22(R)-HC, 24(S)-HC, and 24,25(S)-epoxycholesterol
demonstrating the highest affinities for LXRs (15, 16, 22). In
contrast, 25-HC is the most potent known suppressor of
SREBP-2 in the regulation of cholesterol synthesis (42). The
cause for different affinities of oxysterols in the regulation ofcholesterol catabolism and synthesis in response to the samecholesterol accumulation is currently unknown. Further investigation to seek out other ligands for LXR could lead to the discovery of a common and real physiological signaling molecule forcholesterol metabolism. Meanwhile, our current study suggeststhat the expression level of LXRs, in addition to their ligand
concentrations, can be another potential method for the regulation of LXR target genes.
It is noteworthy that in some experiments, RXR and its ligand
exhibited an effect similar to or even higher than those of LXRs
and their ligands on SREBP-1c activation. Since RXR forms
heterodimers with different nuclear receptors such as retinoic acidreceptor, peroxisome proliferator-activated receptors, farnesoid
X receptor, and some orphan receptors, the effect of RXR activation should be diverse. Therefore, it was also possible that analternative heterodimerizing nuclear receptor might be involvedin SREBP-1c activation. Tissue specificity of copartners for RXR
heterodimers might be crucial for the physiological consequencesof RXR manipulation. SR⫽BP-1c seems to be involved in energy disturbances causing fatty liver, remnant particle production, and insulin resistance and may therefore be a therapeutictarget for these fuel metabolism disorders. Together with re-cent reports on the activation of ABC1 as a reverse cholesteroltransporter, LXR-RXR could be a useful tool with clinicalapplication in the control of cholesterol and fuel metabolic
disorders.
The current studies raise the possibility of a novel link between lipogenesis and sterol regulation through SREBP-1c
and LXR-RXR. Its physiological relevance should be tested indifferent tissues in vivo. Investigation of the involvement of the
LXRE complex in the nutritional regulation of SREBP-1cshould be investigated. It might open up a new aspect of theroles of oxysterols and a novel link between lipogenesis andsterol regulation through SREBP-1c and LXR-RXR.
ACKNOWLEDGMENTS
We thank N. Emoto and A. Amemiya for great help in constructionof the expression library.
This study was supported by the Promotion of Fundamental Studies
in Health Science of the Organization for Pharmaceutical Safety andResearch and health sciences research grants (Research on Human
Genome and Gene Therapy) from the Ministry of Health and Welfare.

ADDENDUM
During the manuscript review process, activation of SREBP1c expression by LXRs was reported in studies using a pharmacological LXR agonist as well as mice deficient in LXR␣,
LXR␤, or both (25a, 28a). The researchers also studied the
SREBP-1c promoter and found one of the LXREs that we
identified in the current study. Their data, from a different
approach to LXRs, and our present SREBP-1c promoter analysis data are basically consistent and confirm each other.
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