A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes

A liver X receptor and retinoid X receptor heterodimer mediates

apolipoprotein E expression, secretion and cholesterol homeostasis

in astrocytes

Yu Liang,*,1 Suizhen Lin,*,1 Thomas P. Beyer,* Youyan Zhang,* Xin Wu,* Kelly R. Bales,* Ronald

B. DeMattos,* Patrick C. May,* Shuyu Dan Li,* Xian-Cheng Jiang,� Patrick I. Eacho,* Guoqing

Cao* and Steven M. Paul*

*Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, Indiana, USA

�State University of New York (SUNY) Downstate Medical Center, Brooklyn, New York, USA

Abstract

Apolipoprotein E (apoE) is an important protein involved in

lipoprotein clearance and cholesterol redistribution. ApoE is

abundantly expressed in astrocytes in the brain and is closely

linked to the pathogenesis of Alzheimer’s disease (AD). We

report here that small molecule ligands that activate either liver

X receptors (LXR) or retinoid X receptor (RXR) lead to a dra-

matic increase in apoE mRNA and protein expression as well

as secretion of apoE in a human astrocytoma cell line (CCF-

STTG1 cells). Examination of primary mouse astrocytes also

revealed significant induction of apoE mRNA, and protein

expression and secretion following incubation with LXR/RXR

agonists. Moreover, treatment of mice with a specific synthetic

LXR agonist T0901317 resulted in up-regulation of apoE

mRNA and protein in both hippocampus and cerebral cortex,

indicating that apoE expression in brain can be up-regulated by

LXR agonists in vivo. Along with a dramatic induction of ABCA1

cholesterol transporter expression, these ligands effectively

mediate cholesterol efflux in both CCF-STTG1 cells and mouse

astrocytes in the presence or absence of apolipoprotein AI

(apoAI). Our studies provide strong evidence that small mole-

cule LXR/RXR agonists can effectively mediate apoE synthesis

and secretion as well as cholesterol homeostasis in astrocytes.

LXR/RXR agonists may have significant impact on the patho-

genesis of multiple neurological diseases, including AD.

Keywords: ABC1, apolipoprotein E, astrocyte, cholesterol

efflux, liver X receptor, retinoid X receptor.

J. Neurochem. (2004) 88, 623–634.

ApoE is an apolipoprotein that plays a critical role in

mediating hepatic clearance of chylomicron remnants, very

low-density lipoproteins (VLDL) and a subclass of high-

density lipoprotein (HDL) particles primarily through its

interaction with the low-density lipoprotein receptor (LDLR)

(Mahley 1988). It is also a molecule that can mediate

cholesterol redistribution in a variety of tissues. Liver is the

primary organ that synthesizes apoE, generating about 70%

of the total body apoE. In most mammals, approximately

20% of the body’s apoE is made in the brain, primarily by

astrocytes, and the remainder is synthesized in other

peripheral tissues (Williams et al. 1985; Mahley 1988).

Macrophages also synthesize and secrete apoE, although at

very low amounts (Basu et al. 1981, 1982). ApoE produced

in macrophages, however, exerts dramatic protection against

atherosclerosis induced by hypercholesterolemia, as has been

amply demonstrated by bone marrow transplantation

experiments (Boisvert et al. 1995; Linton et al. 1995). In

humans, three common apoE variants (apoE2, apoE3 and

apoE4) with either cysteine or arginine at amino acids 112

Received August 5, 2003; revised manuscript received September 26,

2003; accepted September 29, 2003.

Address correspondence and reprint requests to Guoqing Cao and

Steven M. Paul, Lilly Research Laboratories, Eli Lilly & Company,

Indianapolis, Indiana 46285, USA. E-mail: [email protected] and

[email protected] Liang and Suizhen Lin contributed equally to this work.

Abbreviations used: AD, Alzheimer’s disease; apoE, apolipoprotein E;

APP, amyloid precursor protein; DIV, days in vitro; FBS, fetal bovine

serum; HDL, high-density lipoprotein; LDLR, low-density lipoprotein

receptor; LXR, liver X receptor; PBS, phosphate-buffered saline; RT,

room temperature; RXR, retinoid X receptor; VLDL, very low-density

lipoprotein.

Journal of Neurochemistry, 2004, 88, 623–634 doi:10.1046/j.1471-4159.2003.02183.x

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 88, 623–634 623

and 158 are present at frequencies of 7.3, 78.3 and 14.3%,

respectively (Hallman et al. 1991).

It has long been recognized that macrophage apoE

expression and secretion is up-regulated following choles-

terol loading (Basu et al. 1981, 1982) and more recently, the

molecular mechanism(s) of this regulation has been shown to

involve a liver X receptor/retinoid X receptor (LXR/RXR)

heterodimer (Laffitte et al. 2001). LXRs were initially

isolated as orphan nuclear receptors that exist in two

different isoforms with distinct tissue distributions (Lu et al.

2001). Certain hydroxylated cholesterols (oxysterols) have

been identified as potential endogenous ligands for these

receptors (Janowski et al. 1996, 1999; Fu et al. 2001).

Disruption of LXRa under hypercholesterolemic conditions

leads to a dramatic accumulation of cholesterol in the liver as

a result of a failure to regulate Cyp7a gene expression/

transcription, which encodes 7a-hydroxylase, the rate-

limiting enzyme that converts cholesterol to bile acid (Peet

et al. 1998). Recently, multiple LXR target genes have been

identified that include ABC cholesterol transporters (Costet

et al. 2000; Repa et al. 2002), lipoprotein modifying

enzymes (Luo and Tall 2000; Cao et al. 2002; Mak et al.

2002a), apolipoproteins (Laffitte et al. 2001; Mak et al.

2002b), a transcription factor (Repa et al. 2000; Schultz

et al. 2000), and various enzymes (Zhang et al. 2001; Joseph

et al. 2002; Cao et al. 2003) involved in lipid and glucose

metabolism. These studies have established LXRs as central

molecules in mediating cholesterol catabolism and glucose

homeostasis in a variety of tissues.

Astrocytes and microglia are the major sources of apoE in

brain with neurons making little to no apoE (Boyles et al.

1985; Pitas et al. 1987). ApoE appears to be an important

apolipoprotein in brain, most likely for redistributing

cholesterol, as it is highly expressed locally and cannot

penetrate the blood brain barrier (Linton et al. 1991). ApoE

expression is significantly induced at sites of neuronal injury,

possibly providing cholesterol necessary for neuronal repair

(Dawson et al. 1986; Ignatius et al. 1986; Snipes et al.

1986). Moreover, local brain cholesterol carried on apoE has

recently been reported to be the rate-limiting factor for

synaptogenesis (Mauch et al. 2001).

Alzheimer’s disease (AD) is a prevalent neurodegenerative

disorder that occurs in 20% of individuals over 60 years of

age. Although the etiology of AD has not been completely

delineated, much evidence has accumulated which strongly

supports a critical role for the amyloid-b peptide (Ab), both

in its soluble and insoluble (fibrillar) forms, in AD patho-

genesis (Hardy and Selkoe 2002). Mutations in the amyloid

precursor protein (APP) gene linked to autosomal dominant

early onset AD have all been shown to predictably alter Absynthesis (Scheuner et al. 1996; Hardy and Selkoe 2002).

The proteolytic products of APP, especially Ab1)42, are

prone to aggregate and are highly toxic to neurons (Hardy

and Selkoe 2002). For familial and sporadic late-onset AD,

genetic epidemiological studies have established that the

apoE4 allele is an important risk factor, increasing the risk of

developing AD from three- to 15-fold (one or two alleles,

respectively) and decreasing the age of onset (Strittmatter

et al. 1993; Strittmatter and Roses 1995). In vitro studies

have demonstrated apoE isoform-dependent binding to Aband to the microtubule-binding protein Tau (Strittmatter et al.

1993, 1994). ApoE has also been reported to stimulate

neurite outgrowth (Nathan et al. 1994; DeMattos et al.

1998), to possess antioxidant properties (Miyata and Smith

1996) and to stimulate cholesterol efflux (Michikawa et al.

2000), all in an isoform-dependent manner. Although our

understanding of apoE CNS biology has improved, the direct

biochemical relationship between apoE and AD remains

unknown. Most importantly, recent in vivo studies in

transgenic mouse models of AD provide convincing evi-

dence that murine and human apoE isoforms can robustly

impact brain Ab deposition, amyloid formation and the

number of neuritic plaques (Bales et al. 1997, 1999;

Holtzman et al. 1999, 2000), all neuropathological hallmarks

of AD. The transgenic mouse studies analyzing the effect of

human apoE expression in astrocytes clearly demonstrate a

beneficial delay in Ab deposition in an isoform and gene-

dosage dependent manner (Holtzman et al. 1999; DeMattos

2002). These data suggest that drugs capable of increasing

human apoE expression in brain may reduce Ab deposition

and amyloid burden and thus slow the progression of AD.

We now provide data for the first time demonstrating that

the LXR/RXR heterodimer regulates apoE expression in

astrocytes. Both native and synthetic small molecule ligands

of LXR or RXR can effectively increase apoE gene

transcription, translation and secretion both in cultured

glial cells and in vivo. These ligands also induce ABCA1

transporter expression and stimulate cholesterol efflux in

astrocytes.

Methods

Animal studies

Eight-week-old C57BL6 mice were purchased from Harlan

Sprague-Dawley (Indianapolis, IN, USA) and acclimated for two

weeks before the experiments. T0901317 (commercially available

from Cayman Chemical, Ann Arbor, MI, USA) was prepared in

wet granules [212.5 mg Povidone, 3.77 g Lactose Anhydrous

(granular) and 64.8 lL Polysorbate 80 (Tween 80) in 250 mL

water]. Animals were treated orally with either vehicle or various

doses of T0901317 once daily or 7 days and killed with CO2

euthanasia. Tissues of different brain regions were dissected and

utilized for both protein and mRNA analysis. Use of mice was

approved by the Institutional Animal Care and Use Committees of

the American Association for Accreditation of Laboratory Animal

Care-accredited institutions and Lilly Research Laboratories in

accordance with the National Institutes of Health Guide for the

Care and Use of Laboratory Animals.

624 Y. Liang et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 88, 623–634

Cell culture

The human astrocytoma cell line CCF-STTG1 was purchased from

ATCC. Cells were maintained in Dulbecco’s modified Eagle’s

medium (DMEM)/F12 (3 : 1) with 10% fetal bovine serum (FBS).

At 80–90% confluency, cells were washed with phosphate-buffered

saline (PBS) and then equilibrated in serum-free medium with 0.2%

fatty acid-free bovine serum albumin (BSA) for 24 h. Cells were

then treated with various reagents in fresh serum-free medium with

0.2% fatty acid-free BSA for various time periods as indicated. At

the end of the treatments, the conditioned media were collected and

the cells were lysed for various analyses.

C57BL6 mouse mixed glial primary cultures were prepared

according to the method described by Petegnief et al. (Petegnief et al.

2001). Cerebral cortices from 1–3-day-old neonatal C57BL/6 mice

were dissected, stripped of their meninges and digested for 30 min

with 6 mL 0.05% trypsin at 37�C. Trypsinization was stopped by

addition of an equal volume of glial culture medium (DMEM: F12

nutrient mixture (3 : 1) plus 10% BSA, penicillin 100 U/mL and

streptomycin 100 lg/mL) with 10 lL deoxyribonuclease I. The

solution was pelleted, resuspended in glial culture medium and

brought to a single cell suspension by repeated pipetting followed by

passage through a 105 lm pore mesh. Cells were seeded onto 24-well

plates at a density of 3 · 105 cell/mL and cultured at 37�C in

humidified 5% CO2-95% air. The medium was replaced by fresh

medium after 6 days in vitro (DIV). Cultures reached confluence at

6–8 DIV and were used between 8 and 12 DIV. Characterization of

these cultures using immunohistochemical staining for astrocytes

(glial fibrillary acidic protein [GFAP]) and microglia (CD11b)

revealed that they contained > 95% astrocytes and fewer than 3%

microglia as previously described (Petegnief et al. 2001).

Northern blotting analysis

Total cellular RNA was isolated by using TriZol reagent (Invitrogen,

Carlsbad, CA, USA). RNA was separated by 1% agarose-MOPS-

formaldehyde gel electrophoresis and transferred to nylon membra-

nes. RNA was then hybridized with various [32P]-labeled cDNA

probes in ExpressHyb (Clontech, Palo Alto, CA, USA). The results were

visualized by X-ray autoradiography. For detecting LXR a and b and

a-tubulin mRNAs, full-length cDNAs were used. For human and mouse

apoE mRNA blotting, polymerase chain reaction (PCR) amplified

fragments were used (human: 5¢-GGCTGCGTTGCTGGTCACA-

TTC and 5¢-ACCGGGGTCAGTTGTTCCTCCA; mouse: 5¢-TGAA-

CCGCTTCTGGGATTAC and 5¢-GTTCCTCCAGCTCCTTTTTG).

Nuclear run-on analysis

The nuclei of CCF-STTG1 cells were isolated according to the

previously described procedure (Schibler et al. 1983). The elongation

reaction was carried out as described (Goldman et al. 1985). DNA

probes were denatured in 0.1 N NaOH for 30 min at room tempera-

ture, neutralized in 6· saline sodium citrate buffer (SSC), and applied

to Hybond-N membranes (10 lg per slot; Amersham Biosciences,

Piscataway, NJ, USA) using a slot-blot apparatus. 32P-labeled RNA

(1–4 · 106 cpm/mL) was hybridized to the membranes in a buffer

containing 10 mM HEPES, pH 7.5, 10 mM EDTA, 0.3 M NaCl, 1%

SDS, 1· Denhardt’s (0.02% polyvinylpurrolidone, 0.02% Ficoll,

0.02% BSA) and 250 lg/mL tRNA at 45�C for 24 h. Membranes were

washed four times for 5 min each in 2·SSC at room temperature, incu-

bated in 2· SSC containing 10 lg/mL RNaseA for 30 min at 37�C,

then washed twice for 30 min each in 0.5·SSC, 0.1% sodium dodecyl

sulfate (SDS) at 65�C. The signal was detected by autoradiography,

and quantitated by a phosphorimager (Fuji, Stamford, CT, USA).

Real-time PCR analysis

Total RNA was isolated from hippocampal and cortical tissue using

the Invitrogen Total RNA Purification System. PCR primers and

probe for mouse apoE mRNA were designed using Primer Express

1.0 software program (Perkin-Elmer, Boston, MA, USA). Sequences

for the forward primer, reverse primer and probe are: 5¢-GCCG-

TGCTGTTGGTCACA-3¢, 5¢-TGATCTGTCACCTCCGGCTC-3¢,6FAM-CCTCGGCTAGGCATCCT GTCA GCA-TAMRA. Primers

for rodent GAPDH primers including the probe were purchased from

PE Biosystems (Foster City, CA, USA). A 1 lg aliquot of total

RNA was reverse transcribed using the SUPERSCRIPT Preampli-

fication System for First Strand cDNA Synthesis (Gibco BRL,

Rockville, MD, USA). Samples without the reverse transcription

superscript enzyme served as negative controls. PCR cycling

conditions were 2 min at 50�C, 10 min at 95�C, followed by 40

cycles at 95�C for 15 s and 60�C for 1 min in a PE-Applied

Biosystems SDS7700 sequence detection system. Each sample was

run in triplicate and the relative mRNA level was calculated using a

standard curve following normalization with GAPDH. Data repre-

sent the mean ± SEM of triplicate values after normalization.

Western blot analysis

Cell lysates and conditioned media were subjected to sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

and then transferred onto polyvinylidene difluoride (PVDF) mem-

branes. After blocking with 5% non-fat milk in TBST (Tris-buffered

saline with 0.1% Tween-20), the membranes were incubated with

antibodies against either human (Chemicon International, Temecula,

CA, USA) or mouse apoE (Biodesign, Saco, Maine, USA), or anti-

b-actin antibody (Sigma, St Louis, MO, USA) at concentrations

suggested by the manufacturers. The membranes were then

incubated with horseradish peroxidase (HRP)-labeled secondary

antibodies, after which the results were visualized using ECL

reagents (Amersham, Piscataway, NJ, USA) and autoradiography.

ELISA analysis for human apoE

Human apoE in conditioned medium was measured by double-

sandwich ELISA (Starck et al. 2000). Ninety-six-well plates were

coated with anti-human apoE monoclonal antibody (Chemicon)

diluted 1 : 500 in PBS for overnight, and then blocked with 2%

BSA with 7.5 g/L glycine in PBS for 2 h at room temperature (RT).

The plates were then washed once in PBS/Tween-20. Samples

(medium and cell lysates) and human apoE standards (Biodesign)

were loaded into wells for 1 h at 37�C. The plates were then washed

(three times) and biotinylated goat anti-human apoE (Biodesign)

1 : 10 000 in dilution buffer applied for 1 h at RT. Streptavidin/HRP

(Amersham) 1 : 5000 was added to the well for 1 h at RT after

thorough washing (three times). After TMB/HRP substrate was

applied and the reaction stopped by adding H2SO4, the result was

read at 450 nm with a plate reader.

Immunofluorescent staining for intracellular apoE

CCF-STTG1 cells were cultured on poly D-lysine-coated 8-well glass

slides and treated with LXR/RXR agonists for up to 72 h. Cells were

LXR regulates apoE expression in astrocytes 625


then fixed with 4% paraformaldehyde in 4% sucrose/PBS for 5 min.

After washing with 1% triton-100X/PBS (three times, 5 min each),

anti-human apoE antibody (biotinylated 1 : 2000, Chemicon) was

applied for 1 h at 37�C. Fluorescence-labeled avidin (1 : 100; Vector

Laboratories, Burlingame, CA, USA) was added for an additional

1 h. Following washing (three times, 5 min each), slides were

then mounted with fluorescent mounting medium containing

4’,6-diamidino-2-phenylindole (DAPI). Cells were visualized and

images were taken using a fluorescence imaging system (Leica with

program from SPOT RT color Diagnostic, Edmond, OK, USA).

Cholesterol efflux

CCF-STTG1 cells were plated in 24-well plates at a concentration of

5 · 105 cells per well and grown for 24 h. Cells were loaded with

50 lg/mL Ac-LDL (Intracel, Issaquah, WA, USA) and 0.5 lCi/mL3H cholesterol (Amersham) in DMEM supplemented with 1%

L-glutamine, 2% glucose and 0.2% fatty acid-free BSA for 24 h.

Cells were washed twice with DMEM supplemented with 1%

L-glutamine, 2% glucose and 0.2% fatty acid-free BSA. The cells

were equilibrated in this medium for 24 h. Cells were again washed

twice with DMEM supplemented with 1% L-glutamine, 2% glucose,

and 0.2% fatty acid-free BSA. Cells were then treated with LXR/

RXR agonists in 0.5% dimethylsulfoxide (DMSO) for 48 h with or

without 20 lg/mL apoA1 (Intracel). Medium was collected and

centrifuged at 16 100 g for 10 min to remove debris. Cells were

lysed with 500 lL 0.2 N NaOH. Radioactivity in the medium and

cells was measured using a scintillation counter (Packard, Meriden,

CT, USA). The percentage efflux was determined for each well

using the formula: counts media/(counts cells + counts media) ·100.

Results

A recent study suggested that 25-hydroxycholesterol could

increase apoE secretion in a human astrocytoma cell line

[CCF-STTG1 cells; (Gueguen et al. 2001)]. 25-Hydroxy-

cholesterol has dual physiological functions. It is a potent

inhibitor of cholesterol biosynthesis and LDL receptor

activity through inhibition of the sterol regulatory element

binding protein (SREBP) (Brown and Goldstein 1999). It is

also a partial agonist of LXR with a reported EC50 in the low

micromolar range (Janowski et al. 1999). The potency of

25-hydroxycholesterol in inducing apoE secretion suggested

that this regulation was primarily mediated through LXR. As

a first step in investigating this possibility, we examined both

LXRa and LXRb mRNA expression in this cell line.

Northern blot analysis indicated that the mRNAs for both

receptors were expressed in CCF-STTG1 cells, but with

significantly more LXRb mRNA being expressed (Fig. 1a).

This observation is consistent with the finding that LXRa is

primarily expressed in the liver, kidney, intestines and

adipocytes while LXRb is ubiquitously expressed (Lu et al.

2001). We next treated CCF-STTG1 cells with a native

ligand (22-(R)-hydroxycholesterol) (Janowski et al. 1999) or

a specific synthetic ligand (T0901317) of LXR (Schultz et al.

2000), as well as a native RXR ligand (9-cis-retinoic acid)

(Mangelsdorf and Evans 1995), for 48 h and measured the

apoE content in both the medium and cells using western blot

analysis. All three ligands dramatically increased apoE

secretion in the medium and protein in the cells (Fig. 1b).

We observed a concentration-dependent (10–1000 nM)

induction of apoE expression following incubation with

(a)

(b)

(c)

Fig. 1 LXR/RXR heterodimer mediated apoE expression in human

astrocytoma CCF-STTG1 cells. (a) Expression of LXRa and b in CCF-

STTG1 cells demonstrated by northern blot analysis. (b) LXR/RXR

heterodimer mediated apoE expression in CCF-STTG1 cells by

western blot analysis. Cells were treated as indicated in the figure for

48 h (10 lM each of 22-(R)-hydroxycholesterol and 9-cis-retinoic acid),

after which western blot analysis was carried out as described in

Methods. (c) LXR/RXR functions as a permissive heterodimer in

mediating apoE expression in CCF-STTG1 cells. Cells were treated

with either a natural ligand of LXR, 22-(R)-hydroxycholesterol (10 lM)

or a native ligand of RXR, 9-cis-retinoic acid (10 lM), or with a com-

bination of both reagents for 48 h, after which the conditioned medium

was collected and subjected to an apoE ELISA as described in

Methods.

626 Y. Liang et al.


T0901317, and this synthetic LXR agonist was considerably

more effective than the native ligand 22-(R)-hydroxycholes-

terol. 9-cis-Retinoic acid was also a potent inducer of apoE

expression and secretion in CCF-STTG1 cells (Fig. 1b).

Interestingly, the increase in apoE protein appeared to be

more prominent in the cell lysate than in the culture medium

at the 48 h time point. By western analysis, apoE appeared in

the cell lysate as a doublet, possibly reflecting a variable

amount of glycosylation.

Since LXR/RXR usually functions as a permissive

heterodimer, we treated CCF-STTG1 cells with 22-(R)-

hydroxycholesterol, 9-cis-retinoic acid, or in combination.

Measurement of apoE in the medium with an ELISA

demonstrated that either agonist alone is able to increase

apoE secretion and that the combination of both resulted in

an additive effect on apoE secretion. These data suggest that

LXR and RXR act as permissive heterodimers in regulating

apoE expression in human astrocytoma cells (Fig. 1c).

We then examined the concentration- and time-dependent

nature of apoE expression/secretion from CCF-STTG1 cells

induced by T0901317. Cells were treated with various

concentrations of T0901317 for up to 48 h and apoE was

measured in both the cell lysate and medium by western blot

analysis. An increase in apoE expression was obvious, even

at the lowest concentrations of T0901317 examined, and

increased approximately five- to 10-fold over control levels

at the highest concentration tested (3 lM) (Fig. 2a). Similar

results were obtained when apoE was measured by ELISA

(Fig. 2b). The basal level of apoE was about 350 ng/mL in

the medium and 80 ng/mg protein in the cell lysate.

Treatment of cells with T0901317 at the highest concentra-

tion tested (3 lM) led to an increase in apoE to approxi-

mately 2400 ng/mL in the medium (about a seven-fold

increase) and 450 ng/mg in the cell lysate (about a six-fold

increase). The calculated EC50 for T0901317 was approxi-

mately 37 nM, which agrees well with the compound’s

reported potency in activating LXR (Fig. 2b) (Schultz et al.

2000). The secretion of apoE into the medium became

prominent approximately 48 h after addition of T0901317 to

the cultures. However, a dramatic increase in apoE protein

within the cells was apparent 8 h after treatment (Fig. 2c).

The induction of intracellular apoE was confirmed using

immunohistochemistry to study apoE expression in these

cells. Compared to control (DMSO-treated) cells, cells

treated with T0901317 had significantly increased expres-

sion of intracellular apoE, which was primarily perinuclear

in nature (Fig. 2d).

The LXR/RXR heterodimer is known to regulate gene

expression transcriptionally. To explore the mechanisms

underlying apoE expression in astrocytes, we measured

steady state mRNA levels in CCF-STTG1 cells treated with

various LXR/RXR ligands (Fig. 3a). 25-Hydroxycholesterol

and 22-(R)-hydroxycholesterol treatment resulted in a

2.3- and 5.7-fold increase in apoE mRNA, respectively,

(a)

(b)

(c)

(d)

Fig. 2 Dose- and time-dependent apoE regulation by LXR agonist

T0901317 in human astrocytoma CCF-STTG1 cells. (a) and (b) LXR

agonist T0901317 induced apoE expression in CCF-STTG1 cells in a

concentration-dependent manner. Cells were treated with T0901317

at the indicated concentrations for 48 h. Conditioned media and cell

lysates were subjected to western blot analysis (a) and ELISA (b) as

described in Methods. The EC50 was calculated by SigmaPlot 8.0,

Regression Wizard. (c) Time course of apoE expression in CCF-

STTG1 cells following exposure to T0901317 (1 lM). ApoE was

measured by western blot analysis. (d) Immunofluorescent studies of

apoE expression induced by the LXR agonist T0901317. Cells were

incubated with T0901317 (300 nM) for 24, 48 and 72 h. Cells were

then fixed and apoE was visualized by immunofluorescence as des-

cribed in Methods. The blue fluorescence indicates nuclear staining

(DAPI) and the green fluorescence represents intracellular apoE. Note

marked increase in perinuclear apoE staining following treatment with

T0901317.



while T0901317 elevated mRNA about 10-fold. 9-cis-

Retinoic acid treatment resulted in an 11-fold increase in

apoE mRNA. The combination of the LXR ligands 22-(R)-

hydroxycholesterol, 25-hydroxycholesterol or T0901317

with 9-cis-retinoic acid led to a further increase in mRNA

levels (22-, 25- and 16-fold increase, respectively). By

contrast, oxysterol 22-(S)-hydroxycholesterol, which does

not activate LXR, did not induce apoE mRNA expression in

CCF-STTG1 cells. To further confirm that the observed

elevation of apoE mRNA was a result of elevated apoE gene

transcription, we conducted nuclear run-on experiments. As

expected, treatment of CCF-STTG1 cells with various LXR/

RXR ligands resulted in a significant increase in apoE

transcription rate (Fig. 3b). The increase in the transcrip-

tional rate paralleled the increase in apoE mRNA levels.

Collectively, these data suggest that LXR/RXR agonists

effectively increase apoE gene transcription, protein synthe-

sis and secretion in human astrocytoma (CCF-STTG1) cells.

We then used fetal mouse primary astrocyte cultures to

investigate whether similar findings are observed in murine

astrocytes. Fetal mouse astrocytes were prepared and treated

with either vehicle or various concentrations of T0901317,

and the medium was examined by western blot analysis for

apoE expression using an antibody that specifically recog-

nizes mouse apoE. We observed a significant increase in

apoE expression in murine astrocytes treated with increasing

concentrations of T0901317 (Fig. 4a). The magnitude of

apoE expression induced by T090137 was, however, less

robust than that observed in the human astrocytoma cell line.

Northern blot analysis of apoE mRNA yielded qualitatively

similar results as in human astrocytoma cells, suggesting a

similar mechanism(s) of action (Fig. 4b).

To examine whether LXR agonists induce apoE expres-

sion in vivo, we treated C57BL6 mice with T0901317 at

doses of 1, 10 and 50 mg/kg (orally) for 7 days. The

hippocampus and cerebral cortex were carefully dissected,

and protein extracts were prepared and subjected to western

blot analysis. A representative western blot of apoE in the

hippocampus is shown in Fig. 5(a). A dose-dependent

increase in apoE protein expression was evident in the

treated groups compared to vehicle. RNA was also prepared

from the contralateral hippocampi, and real-time PCR

(b)

(a)

Fig. 3 LXR/RXR heterodimer mediates apoE expression in human

astrocytoma cells by increasing transcription. (a) Northern blot ana-

lysis of apoE mRNA in CCF-STTG1 cells treated with various LXR or

RXR ligands for 48 h (see figure for details). Northern blot analysis

was carried out as described in Methods. (b) Nuclear run-on analysis

of LXR/RXR mediated apoE expression in CCF-STTG1 cells. Cells

were treated with various LXR/RXR ligands and the nuclei were pre-

pared for run-on studies as described in Methods. KS is a negative

control and GAPDH is a control utilized to normalize sample loading.

Note the parallel increase in the rate of apoE transcription and apoE

mRNA levels following various compound treatments.

(a)

(b)

Fig. 4 LXR/RXR heterodimer mediates apoE expression in mouse

fetal primary astrocytes. Fetal mouse primary astrocyte cultures were

prepared as described in Methods. The cells were treated with

T0901317 at the indicated concentrations for 48 h. The medium was

collected and subjected to western blot analysis (a) with a specific anti-

mouse apoE antibody as described in Methods. Total RNA was pre-

pared from cells treated with different LXR and/or RXR ligands for

48 h. Northern blot analysis was carried out to evaluate apoE mRNA

expression (b). Note the increase in apoE protein and mRNA induced

by T0901317.

628 Y. Liang et al.


analysis was performed to measure apoE mRNA. A statis-

tically significant increment in apoE was detected when

higher doses (10 and 50 mg/kg) of T0901317 were used

(Fig. 5b). In the cerebral cortex, a similar effect on apoE

mRNA expression was observed, with a modest but signi-

ficant increase in apoE protein when 50 mg/kg T0901317

was administered (Figs 5c and d).

The LXR/RXR heterodimer is known to modulate

ABCA1 transporter expression and activity that is rate-

limiting in cholesterol efflux in cells from peripheral tissues.

As cellular cholesterol is closely linked to Ab metabolism,

we examined ABCA1 expression in CCF-STTG1 cells.

Treatment of cells with T0901317 resulted in a concentra-

tion-dependent increase in ABCA1 mRNA (Fig. 6a) with a

calculated EC50 of 24.4 nM. As expected, 22-(S)-hydroxy-

cholesterol had very little effect on ABCA1 mRNA, while

treatment with 22-(R)-hydroxycholesterol or 25-hydroxy-

cholesterol had a dramatic effect on inducing ABCA1

mRNA expression. Interestingly, 9-cis-retinoic acid, a known

ligand of RXR, exhibited only a minimal effect on ABCA1

Fig. 6 LXR/RXR heterodimer mediates ABCA1 expression in astro-

cytoma cells. CCF-STTG1 cells were treated with LXR or/and RXR

ligands for 24 h and ABCA1 mRNA was analyzed by the branched

DNA method (Zhang et al. 2002). (a) Treatment of CCF-STTG1 cells

with T0901317 resulted in a concentration-dependent induction of

ABCA1 mRNA. (b) ABCA1 expression is induced by a variety of LXR

and/or RXR ligands.

(a)

(b)

(c)

(d)

Fig. 5 In vivo regulation of apoE expression in the hippocampus (a, b)

and the cerebral cortex (c, d) by the LXR agonist T0901317. C57B6

mice (n ¼ 6) were treated orally by gavage either with vehicle (see

Methods) or with 1, 10 and 50 mg/kg of T0901317 for 7 days. Animals

were killed at the end of the study and different brain regions were

carefully dissected for western blot analysis (a, c) or real-time PCR

analysis (b, d) as described in Methods. Western blot data in (a) and

(c) are representative blots from a single animal in each treatment

group (n ¼ 6), where GFAP immunoreactivity was utilized as a sample

loading control. Bar graphs in (a) and (c) represent the quantitative

western blot data (apoE/GFAP) from all animals (n ¼ 6). *p < 0.05;

**p < 0.01. Unpaired t-test was used for statistical analysis.



mRNA. However, combined treatment of cells with LXR and

RXR ligands resulted in an additive effect on ABCA1

mRNA level (Fig. 6b). The regulation of these important

gene products involved in cholesterol metabolism strongly

suggests that LXR/RXR agonists can effectively mediate

cholesterol homeostasis in astrocytes. We therefore measured

cholesterol efflux in both CCF-STTG1 cells and mouse

primary astrocytes treated with various LXR/RXR ligands in

the presence or absence of the cholesterol acceptor apolipo-

protein AI (apoAI). Cells were pre-loaded with radiolabeled

cholesterol and then treated with various concentrations of

LXR/RXR ligands. Cell viability was closely monitored and

no apparent toxicity was observed with various LXR/RXR

ligand treatments. In CCF-STTG1 cells, the addition of

apoAI (20 lg/mL) into the medium alone resulted in a

significant increase (68%) in cholesterol efflux, suggesting

that these cells have a relatively high capacity for cholesterol

efflux. Treatment of the cells with increasing concentrations

of T0901317 resulted in a further increase in cholesterol

efflux in the presence of apoAI (maximum increase of 180%

at 10 lM). Treatment with 9-cis-RA yielded similar results,

while combining both ligands led to a maximal increase in

cholesterol efflux (368%) when apoAI was present in the

medium. Notably, even in the absence of apoAI, treatment of

the cells with T0901317 (10 lM) resulted in a significant

increase in cholesterol efflux (43%), suggesting that endog-

enously produced apoE might be sufficient to mediate

cholesterol efflux, either by directly facilitating the process

of cholesterol secretion or by functioning as a cholesterol

acceptor. Treatment with 9-cis-RA also resulted in a 226%

increase in cholesterol efflux in the absence of apoAI, while a

combination of T0901317 and 9-cis-RA further augmented

cholesterol efflux (Fig. 7a). Similar results were obtained in

mouse primary astrocytes (Fig. 7b). The addition of apoAI

into the medium resulted in a 235% increase in cholesterol

efflux. Treatment with T0901317 or 9-cis-RA induced

significant increases in cholesterol efflux in the presence or

absence of apoAI. Taken together, these results strongly

indicate that LXR/RXR ligands can effectively modulate

expression of critical target genes involved in lipoprotein

metabolism and mediate cholesterol homeostasis in astro-

cytes.

Discussion

Alzheimer’s disease is a neurodegenerative disorder that

affects approximately 20% of the population over 60 years of

age. Although the pathogenesis of AD has not been

completely defined, considerable genetic and biochemical

data have accumulated supporting the b-amyloid hypothesis

of AD, which posits a critical role for the proteolytic

processing products of the b-amyloid precursor protein

(APP), the Ab peptides (Hardy and Selkoe 2002). The

relative levels of brain Ab are determined by its synthesis as

well as its clearance rate. ApoE does not appear to affect Absynthesis in vivo, but can avidly bind Ab and appears to play

a critical role in brain Ab clearance in isoform-dependent

fashion (LaDu et al. 1994; Bales et al. 1997, ; Holtzman

et al. 1999, 2000; DeMattos 2002). Recent studies utilizing

either mouse apoE knockout or human apoE transgenic mice

bred to PDAPP transgenic mice have demonstrated that apoE

is an important determinant of Ab deposition and amyloid

formation in this mouse model of AD (Bales et al. 1997,

1999; Holtzman et al. 1999, 2000; DeMattos 2002). It is

important to note that expression of human apoE, especially

apoE3, reduces both soluble and insoluble brain Ab in a gene

dose-dependent manner (Holtzman et al. 1999; DeMattos

2002). In this context, our observation that small molecule

agonists of LXR/RXR can effectively increase apoE mRNA,

protein synthesis and secretion may have important thera-

peutic implications. The regulation of apoE expression in a

human astrocytoma cell line by LXR/RXR is fairly robust,

and is also observed in mouse primary astrocytes and in

specific brain regions of mice treated with the selective LXR

agonist T0901317. However, the regulation of apoE expres-

sion in mouse astrocytes and brain by LXR/RXR agonists is

Fig. 7 LXR/RXR heterodimer mediates cholesterol efflux in astro-

cytes. (a) Cholesterol efflux studies in CCF-STTG1 cells. Cells were

seeded in 24-well plates and loaded with radiolabeled cholesterol

together with acetylated LDL. Cells were then treated with LXR/RXR

ligands for 48 h with or without exogenously added ApoAI. The per-

cent cholesterol efflux was calculated as described in Methods. (b)

Cholesterol efflux studies in mouse primary astrocytes. Mouse primary

astrocytes were prepared and cholesterol efflux study was conducted

as previously described. Grey and black bars indicate cholesterol

efflux without or with apoAI addition, respectively.

630 Y. Liang et al.


quantitatively rather modest compared with CCF-STTG1

cells. Future studies will extend these findings to apoE

knockin mice with different human apoE isoforms. These

studies should determine whether pharmacological modula-

tion of apoE expression via the LXR/RXR heterodimer alters

Ab metabolism, deposition and amyloid formation. It is

important to note that apoE is but one of the target genes for

LXR/RXR activation, and that other genes involved in lipid

metabolism will be activated by LXR/RXR agonists as well.

Joseph et al. have recently reported that LXR ligands also

can mediate anti-inflammatory actions (Joseph et al. 2003),

and inflammatory processes have been implicated in the

etiology of AD (Rogers et al. 1996).

Recent data have also implicated cholesterol metabolism

in the pathogenesis of AD (Jarvik et al. 1995; Notkola et al.

1998). Although conclusive results have not yet been

obtained with the cholesterol-lowering drugs either in

preventing or treating AD, pre-clinical data strongly suggest

that cholesterol plays a role in Ab metabolism (Sparks et al.

1994; Fassbender et al. 2001; Golde and Eckman 2001;

Kojro et al. 2001). For example, acyl cholesterol acyltransf-

erase (ACAT), the enzyme that converts free cholesterol to its

ester, has been implicated in the processing of APP to Ab(Puglielli et al. 2001). In the present report we show that

apoE expression and secretion induced by LXR/RXR ligands

can effectively increase cholesterol efflux in astrocytes even

in the absence of exogenously added apolipoproteins such as

apoAI. As cholesterol synthesis in brain is believed to be a

much slower process compared to other tissues, modulating

cholesterol efflux as a way of impacting overall cholesterol

homeostasis in brain may represent a more efficient way to

eliminate cholesterol from the CNS. It has been postulated

that the major route of cholesterol efflux from brain is by the

enzymatic hydroxylation of cholesterol via cholesterol

hydroxylases to produce monohydroxylated cholesterol,

which can then freely diffuse through the plasma membrane.

It is interesting to note that hydroxylated cholesterol,

including 25-hydroxycholesterol, 27-hydroxycholesterol

and 24-hydroxycholesterol, are all LXR ligands (Janowski

et al. 1999; Fu et al. 2001). Moreover, 24-cholesterol

hydroxylase is specifically expressed in the brain (Lund

et al. 1999). LXR has been shown to be abundantly

expressed in the brain, especially LXRb with a relatively

higher level of expression than LXRa (Lu et al. 2001). These

data implicate LXRs and their target genes as being

important in brain cholesterol and lipid metabolism. In this

regard, LXR double-knockout mice display severe CNS

abnormalities with significantly increased lipid deposition

and neurodegeneration (Wang et al. 2002).

The exact effect(s) of increasing apoE expression and

secretion as well as cholesterol efflux by astrocytes on APP

processing, Ab synthesis and clearance is still poorly

understood. Fukomoto et al. reported that treatment of

primary neuronal cultures with LXR agonists increased Ab

secretion, while Koldamova et al. observed a decrease in Absecretion (Fukumoto et al. 2002; Koldamova et al. 2003).

Future studies to delineate how increased apoE secretion and

lipidation via LXR/RXR activation impact Ab synthesis and

clearance in vitro and in vivo will be required.

Our results also suggest that LXR/RXR ligands may have

utility in brain or spinal cord repair following injury, as well

as in synaptogenesis (Dawson et al. 1986; Ignatius et al.

1986; Snipes et al. 1986; Mauch et al. 2001). It is interesting

to note that both ABCA1 and apoE are dramatically

up-regulated at sites of brain injury, implicating the induction

of key molecules involved in cholesterol efflux and redistri-

bution in neuronal repair. LXR ligands can effectively

modulate the expression of both target genes, suggesting that

such ligands may greatly facilitate the cholesterol redistri-

bution process. Whether the LXR/RXR heterodimer is

directly involved in the injury-induced expression of apoE

and ABCA1 is not known but is an intriguing possibility. A

recent report has also identified cholesterol carried on apoE

as the rate-limiting factor in synaptogenesis (Mauch et al.

2001), thus suggesting a potential application of activating

LXR in augmenting the process of synaptogenesis. In this

regard, it is important to note that LXR agonists can increase

cholesterol efflux even in the absence of exogenously added

cholesterol acceptor. These findings suggest that apoE can

effectively function as a cholesterol acceptor in mediating

cellular cholesterol efflux. It will be interesting to investigate

whether increased apoE secretion and lipidation induced by

LXR/RXR agonists can lead to augmented neurite outgrowth

as a result of increased lipid/cholesterol delivery.

Cellular cholesterol homeostasis is achieved through a fine

balance of synthesis, uptake and efflux. Recently it has been

shown that mutations in the ATP binding cassette transporter

(ABCA1) gene constitute the molecular defect in Tangier

disease, a rare genetic disorder manifested by defects in the

cholesterol efflux process mediated by apolipoproteins

resulting in a nearly complete absence of HDL cholesterol

(Bodzioch et al. 1999; Brooks-Wilson et al. 1999; Lawn

et al. 1999; Rust et al. 1999). Active cholesterol efflux may

very likely involve the interplay between ABCA1 and a

cholesterol acceptor such as apoAI that is in close proximity

to ABCA1. While the cholesterol efflux process has been

extensively characterized in cells from the periphery, little is

known about the cholesterol efflux process in brain. ApoE as

the major apolipoprotein in brain may play a central role in

this process, although apoAI has also been reported to be

present in cerebrospinal fluid (Roheim et al. 1979). ApoE is

abundantly secreted and generates HDL-like lipoprotein

particles in the medium of mouse primary astrocytes (Pitas

et al. 1987). Fagan et al. showed that apoE is essential for

this process (Fagan et al. 1999). It has also been shown that

apoE can act as cholesterol acceptor either when added

exogenously or produced intracellularly in peripheral cells

such as macrophages (Smith et al. 1996; Lin et al. 1999;



Remaley et al. 2001). Here, we show that in astrocytes,

pharmacological induction of apoE expression, along with

ABCA1 expression and potentially other target genes

involved in lipid homeostasis, is sufficient to increase

cholesterol efflux without adding an exogenous choles-

terol acceptor. In this regard, Michikawa et al. (Michikawa

et al. 2000) reported that cholesterol efflux in neurons

mediated by apoE is isoform-dependent (E2 > E3 > E4),

which may partly explain the finding that apoE4 is associated

with late onset and sporadic AD (Michikawa et al. 2000;

Gong et al. 2002). Endogenously produced apoE is more

efficient in mediating cholesterol efflux in astrocytes (Ito

et al. 1999; Gong et al. 2002). Taken together, these data

suggest that increasing brain apoE expression may prevent or

treat AD.

LXR has been identified as master transcription factor

regulating lipid and glucose metabolism (Edwards et al.

2002). We have shown here that activation of LXR in

astrocytes may potentially impact brain apoE expression and

other target genes to mediate brain cholesterol homeostasis.

The apoE gene was previously reported to be regulated by

LXR in both human and murine macrophages, which was

attributed to a pair of LXR responsive elements (LXRE)

identified at the 3¢ end of the human apoE gene in a region

called the multi-enhancer (ME) region that has been shown

to be essential for apoE expression in macrophages and

adipocytes (Shih et al. 2000; Laffitte et al. 2001). Although

Whitney et al. failed to detect significant changes in apoE

expression in brain following LXR activation in vivo

(Whitney et al. 2002), we have observed significant induc-

tion of both apoE mRNA and protein in human astrocytoma

cells, murine primary astrocytes, and in the mouse hippo-

campus and cerebral cortex in vivo. The reason(s) for this

discrepancy is unknown. However, Taylor and colleagues

have recently identified ME regions of the human apoE gene

essential for human apoE gene expression in astrocytes

(Grehan et al. 2001). We speculate that LXR regulation of

apoE expression in astrocytes may require similar cis-

elements as in macrophages. The molecular mechanism of

the differential response of the apoE gene to LXR/RXR

ligands in human cells and in mouse primary astrocytes is

also not clear at the present time. Possible explanations

include duplication of the ME region in the human apoE

gene, differences in the flanking regions surrounding LXRE,

and differences between transformed cells and those charac-

teristic of primary cultures.

In summary, we have identified apoE as a target gene for

the LXR/RXR heterodimer in human and mouse astrocytes,

and demonstrate that activation of the LXR/RXR heterodi-

mer can effectively mediate cholesterol homeostasis in

astrocytes. Our results suggest that treatment with small

molecule LXR/RXR agonists may represent a potential

therapeutic approach to AD and other neurodegenerative

disorders.

Acknowledgements

We would like to thank Drs Steve Kuolong Yu and Timothy Grese

for making the compound available for the study. We also thank Su

Wu and Deanna Webb for technical assistance.

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A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes