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Endocrinology Vol. 143, No. 7 2548-2558
Copyright © 2002 by The Endocrine Society

RECEPTORS

A Novel Liver X Receptor Agonist Establishes Species Differences in the Regulation of Cholesterol 7{alpha}-Hydroxylase (CYP7a)

John G. Menke, Karen L. Macnaul, Nancy S. Hayes, Joanne Baffic, Yu-Sheng Chao, Alex Elbrecht, Linda J. Kelly, My-Hanh Lam, Azriel Schmidt, Soumya Sahoo, Jianhua Wang1, Samuel D. Wright, Patrick Xin, Gaochao Zhou, David E. Moller and Carl P. Sparrow

Molecular Endocrinology (J.G.M., K.L.M., N.S.H., L.J.K., P.X., G.Z., D.E.M.), Atherosclerosis and Endocrinology (J.B., Y.-S.C., M.-H.L., S.D.W., C.P.S.), Bioinformatics (A.E.), Medicinal Chemistry (S.S.) at Merck \|[amp ]\| Co., Rahway, New Jersey 07065; and Bone Biology (A.S.) at Merck & Co., West Point, Pennsylvania 19486

Address all correspondence and requests for reprints to: John Menke, Merck Research Laboratories, Department of Molecular Endocrinology, RY80W-207, 126 East Lincoln Avenue, Rahway, New Jersey 07065. E-mail: . John_Menke{at}merck.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The liver X receptors, LXR{alpha} and LXRß, are members of the nuclear receptor superfamily. Originally identified as orphans, both receptor subtypes have since been shown to be activated by naturally occurring oxysterols. LXR{alpha} knockout mice fail to regulate cyp7a mRNA levels upon cholesterol feeding, implicating the role of this receptor in cholesterol homeostasis. LXR activation also induces the expression of the lipid pump involved in cholesterol efflux, the gene encoding ATP binding cassette protein A1 (ABCA1). Therefore, LXR is believed to be a sensor of cholesterol levels and a potential therapeutic target for atherosclerosis. Here we describe a synthetic molecule named F3MethylAA [3-chloro-4-(3-(7-propyl-3-trifluoromethyl-6-(4,5)-isoxazolyl)propylthio)-phenyl acetic acid] that is more potent than 22(R)-hydroxycholesterol in LXR in vitro assays. F3MethylAA is capable not only of inducing ABCA1 mRNA levels, but also increasing cholesterol efflux from THP-1 macrophages. In rat hepatocytes, F3MethylAA induced cyp7a mRNA, confirming conclusions from the knockout mouse studies. Furthermore, in rat in vivo studies, F3MethylAA induced liver cyp7a mRNA and enzyme activity. A critical species difference is also reported in that neither F3MethylAA nor 22(R)-hydroxycholesterol induced cyp7a in human primary hepatocytes. However, other LXR target genes, ABCA1, ABCG1, and SREBP1, were regulated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LIVER X RECEPTORS (LXRs) are ligand-activated transcription factors that are members of the family of proteins referred to as nuclear receptors. It is believed that nuclear receptors, when bound by ligands, regulate their target genes by a complex interaction between other nuclear transcription factors, cofactors, and with specific nucleotide sequences, or response elements, in the promoter of these genes. LXRs consist of two closely related protein isoforms encoded by separate genes—LXR{alpha} (1, 2) and LXRß (also termed NER) (3). The rat homologs of these two receptors are RLD-1 and orphan receptor-1, respectively. Both LXR isoforms have recently been shown to be activated by specific oxysterols (4, 5, 6). These studies also demonstrated the structure-function relationship of LXR ligands with respect to the position of the hydroxyl moiety and its stereo conformation (5, 6). As with the peroxisome proliferator-activated receptors (PPARs), LXRs heterodimerize with the retinoid X receptor (RXR) (6, 7). These heterodimers have been described as being permissive in that they can be activated by LXR as well as RXR agonists (1, 8). Nuclear receptors that form heterodimers with RXR have been shown to interact with DNA response elements that are repeats of the core sequence of AGGTCA separated by 1–5 bp (1, 7). LXR-RXR heterodimers have been shown to bind to a direct repeat that is separated by 4 bp (DR4) (6, 7). One of the proposed target genes for LXR is cyp7a, which encodes the enzyme cholesterol 7{alpha}-hydroxylase (CYP7a) (5, 6, 9). Thus, a putative LXR response element was identified in the cyp7a promoter (6). Cyp7a is the rate limiting step in the classical pathway for the conversion of cholesterol to bile acids (10). Therefore, one possible site for the control of cholesterol homeostasis by LXR is the regulation of cholesterol metabolism to bile acids and excretion in the bile. Dysregulated cholesterol homeostasis is clearly responsible for a major proportion of the pathogenesis of atherosclerosis, gall stones, and other lipid storage disorders (11). Previously, a species difference for the induction of cyp7a was suggested. It was reported that mice and rats respond to cholesterol feeding with induction of cyp7a (12). Conversely, rabbits (13) and humans (14) appear to lack this response. This species difference was linked to LXR by EMSAs. Cyp7a LXR response elements (LXREs) from mice and rats were shifted in the presence of LXR{alpha} and RXR, whereas the human cyp7a LXRE was not shifted (15).

Both LXR{alpha} and LXRß knockout mice have recently been described. LXR{alpha} null mice are characterized by their striking inability to maintain normal cholesterol homeostasis when challenged with a high cholesterol diet (9). In contrast, LXRß null mice are notable for their near absence of a phenotype with respect to altered cholesterol metabolism (16). When both LXR{alpha} and LXRß are knocked out, there is an accentuated inability for these mice to cope with a cholesterol challenge when compared with the single LXR{alpha} knockout mice (17). This suggests that LXRß does play a role in cholesterol homeostasis. These studies also suggest that LXR{alpha} is able to compensate for the loss of LXRß, where LXRß is unable to compensate for the loss of LXR{alpha} in mice.

More recently, a potential connection has been made between LXR and regulation of ABCA1, which is now known to be required for the maintenance of high density lipoprotein (HDL) cholesterol levels. The genetic basis of Tangier disease is a mutation in the ABCA1 gene (18, 19, 20, 21). Due to the loss of functional ABCA1 protein, these patients have a decreased ability to efflux cholesterol out of cells and onto apolipoprotein AI (apoAI) to form HDL. Indeed, Tangier disease is characterized by low HDL cholesterol and an increased predisposition to atherosclerosis. Recent evidence suggests that ABCA1 is a direct target gene of LXR (17, 22, 23, 24).

The hypothesis that LXR can sense cholesterol levels and mediate the induction of genes within pathways involved in the removal of cholesterol makes LXR an attractive pharmaceutical target. In addition to the use of knockout mice, the availability of potent synthetic agonists for LXR is a critical aspect required for further validation of LXRs as potential drug targets and to gain a broader understanding of the function of LXR. T0901317, a potent and selective LXR agonist, has been described in the literature, and the biological effects of this agonist have been studied (17, 25). Here we describe the discovery and biological characterization of a novel LXR agonist, 3-chloro-4-(3-(7-propyl-3-trifluoromethyl-6-(4,5)-isoxazolyl)propylthio)-phenyl acetic acid (F3MethylAA), which is structurally distinct from T0901317. Results obtained using this compound support a major role for LXRs in the regulation of both cholesterol and triglyceride metabolism in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Tissue culture reagents and lipofectamine were purchased from Life Technologies, Inc. (Rockville, MD). Human serum type AB, Roswell Park Memorial Institute (RPMI) and antibiotic-antimyotic solutions were received from Sigma (St. Louis, MO). Charcoal-stripped fetal calf serum (CS-FCS) was obtained from Gemini Bio-Products, Inc. (Calabasas, CA). Cell culture lysis buffer and luciferase (luc) assay buffer were purchased from Promega Corp. (Madison, WI). 22(R)-Hydroxycholesterol [(22R)OH cholesterol] was obtained from Steraloids, Inc. (Newport, RI). Taqman probes and primers were purchased from PE Applied Biosystems (Foster City, CA). Both the F3MethylAA and 3-chloro-4-(3-(3-pheny-7-propylbenzofuran-6-yloxy)propylthio)-phenyl acetic acid (PBFuranAA) were synthesized at Merck Research Laboratories (Rahway, NJ).

Human hepatocytes
Human primary hepatocytes were obtained post mortem from In Vitro Technologies (Baltimore, MD) in six-well dishes. Both donors were not overtly ill and whose reported cause of death were cerebrovascular accidents. Cells were allowed to recover overnight in phenol red-free DMEM (high glucose) supplemented with 10% FCS, 1% nonessential amino acids, 1% glutamine, 100 U/ml penicillin G and 100 µg/ml Streptomycin sulfate at 37 C in a humidified atmosphere of 5% CO2. Hepatocytes were treated with the appropriate compound in 0.1% ethanol or 0.1% dimethyl sulfoxide (DMSO) for 24 h in the above media containing 10% CS-FCS followed by application of fresh media and compound for an additional 6 h.

Rat primary hepatocytes
Suspension cultures of rat primary hepatocytes were obtained from Cedra Corp. (Austin, TX), rinsed and plated in Williams Media E containing 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, 0.1 µM dexamethasone and 1 µM T3. Cells were allowed to recover overnight before addition of compounds in fresh media for a total of 48 h.

Monocyte-derived macrophages
Monocytes were isolated from the blood of healthy volunteers as described (26). T cell-depleted suspensions of human monocytes were differentiated into macrophages by the method of Wright and Silverstein (27). Briefly, cells in suspension were cultured in Teflon jars in RPMI 1640 containing 12% human serum, 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate at 37 C in a humidified atmosphere of 5% CO2. After 7–9 d incubation, cells were collected from prechilled jars and centrifuged at 1000 rpm for 12 min at 4 C. Pooled cells were seeded into 12-well plates at a density of 6 x 105 cells per well. Treatment of macrophages was initiated the following day as described above for human primary hepatocytes.

Preparation of recombinant human LXR{alpha} and LXRß
Human LXR{alpha} and LXRß ligand binding domain (LBD) receptors were expressed as glutathione S-transferase (GST)-fusion proteins. The cDNA for human LXR{alpha} (encoding amino acids 164–447) was cloned into the SalI/XhoI sites in the pGEX-4T3 vector (Amersham Pharmacia Biotech, Arlington Heights, IL) and the cDNA for human LXRß (encoding amino acids 149–455) was cloned into the EcoRI site of the pGEX-1{lambda}T vector (Amersham Pharmacia Biotech). Escherichia coli BL-21 cells transformed with the respective plasmids were propagated in Luria Bertani broth (Life Technologies, Inc.) containing 50 µg/ml ampicillin at 37 C to OD600 of 0.6–0.8 before induction at room temperature for 4 h with 0.4 mM isopropylthio-ß-D-galactoside. The cells were harvested by centrifugation at 6000 rpm, 4 C for 10 min and suspended in ice-cold PBS containing 0.5% Triton X-100, 5 mM dithiothreitol (DTT) and 0.5 mM phenylmethylsulfonyl fluoride. The cells were lysed by two passes through an EmulsiFlex-C5 high pressure homogenizer (Avestin, Inc., Ottawa, Ontario, Canada) as per the manufacturer’s operating instructions for homogenizing bacteria. Cellular debris was removed by centrifugation at 15,000 rpm, 4 C for 30 min. Recombinant GST-LXR LBD receptors were purified by affinity chromatography on glutathione sepharose resin as per the manufacturer’s instructions (Amersham Pharmacia Biotech). Receptors were eluted in a buffer of 50 mM Tris, 50 mM NaCl, 5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and 10 mM glutathione. Peak fractions, as determined by Bradford protein analysis, were pooled and glycerol added to a final concentration of 50% to stabilize the receptor protein.

Scintillation proximity assays
As an approach to identify and characterize human LXR ligands, LXR scintillation proximity assays (LXRSPA) were developed. Using the GST-LXR LBD receptors for {alpha} and ß, optimizations were performed for each component of the assay buffer as well as assay conditions to achieve maximal signal-to-noise ratio (~5-fold). Several independent saturation binding analyses using increasing amounts of [3H2]F3MethylAA and an excess of unlabeled F3MethylAA were performed to determine dissociation constants. Nonlinear regression analyses calculated the dissociation constant values for the human GST-LXR{alpha} and LXRß LBD to be 15 nM and 13 nM, respectively (data not shown). Once the assay conditions had been established, competitive binding experiments were performed in Packard OptiPlate-96 well polystyrene microplates (Packard BioScience Co., Meriden, CT). In these assays, a minimal amount of receptor (~5 nM) was used to achieve an acceptable signal. The human GST-LXR LBDs were added to a final volume of 74 µl SPA buffer (10 mM Tris, pH 7.2; 1 mM EDTA; 10% glycerol; 10 mM Na molybdate; 1 mM DTT; and 2 µg/ml benzamidine), 0.1% nonfat dry milk, 8.3 µg/ml anti-GST antibody (Amersham Pharmacia Biotech), and 25 nM [3H2] F3MethylAA (specific activity of 13.4 Ci/mmol). Yttrium silicate protein A-coated SPA beads (Amersham Pharmacia Biotech), suspended in SPA buffer, were added to a final concentration of 1.25 mg/ml. Test compound (1 µl) in DMSO was then added to the assay. Nonspecific binding was determined by addition of 2.5 µM cold F3MethylAA. After incubation for ~16 h at 15 C, with shaking, the assay plates were counted in a TopCount (Packard Instrument Co.) to determine the displacement of radioligand from the receptor by test compounds. Results are expressed as percent inhibition and inflection points calculated by a four-parameter logistic equation. Equilibrium dissociation constants (Kis) are calculated by the equation of Cheng and Prusoff (28).

Transactivation assay
Functional activity of LXR ligands was determined in a cell-based transactivation assay. The chimeric receptor expression construct, phLXR{alpha}/GAL4, was prepared by fusing the human ligand binding domain (LBD) of LXR{alpha} to the DNA binding domain of the yeast transcription factor GAL4. The reporter vector, pUAS(5x)-tk/luc, contains five repeats of the GAL 4 response element (upstream activating sequence) and a minimal thymidine kinase promoter adjacent to luc. The green fluorescence protein plasmid, pEGFP-N1, was obtained from CLONTECH Laboratories, Inc. (La Jolla, CA) and used as a normalizing control. HEK-293 cells were seeded at 4 x 104 cells/well in opaque 96 well cell culture plates in DMEM containing 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin and maintained at 37 C in an atmosphere of 5% CO2 in air. After a 20-h incubation, cells were transfected with 2 ng phLXR/GAL4, 20 ng pUAS(5x)-tk/luc and 34 ng of pEGFP-N1 in the presence of 1.3 µg Lipofectamine (Life Technologies, Inc.) according to the manufacturer’s instructions. Transfection medium was removed after 5 h and replaced with test compounds in DMEM containing 10% CS-FCS, 1% glutamine, 1% nonessential amino acids, and antibiotics. After an additional 44 h, cells were lysed in 50 µl cell culture lysis buffer. Green fluorescent protein activity was measured in a CytoFluor multiwell plate reader (PE Biosystems, Inc.). Luc activity was determined using luc assay buffer (Promega Corp.) in a ML3000 Luminometer (ThermoLabsystems, Beverly, MA). Results are expressed as fold induction compared with DMSO control.

Cofactor association assay
The ligand dependent interaction between LXR{alpha} or LXRß and a fragment of SRC-1 coactivator (steroid receptor coactivator-1) was determined by a homogeneous time-resolved fluorescence assay, similar to that previously described by Zhou et al. (29). The reaction consisted of 198 µl of 50 mM HEPES, 125 mM KF, 0.125% (wt/vol) 3-[3-cholamidopropyl)diethylammonio]-1-propane-sulfonate, 0.05% casein, 5 nM GST-LXR LBD, 2 nM anti-GST-(Eu)K, 10 nM biotin-SRC-1 (amino acids 568–780), 20 nM streptavidin/XL665). To this were added 2 µl of vehicle or test compound. Following overnight incubation at 4 C, fluorescence measurements were made using the Packard Discovery. Results are expressed as the ratio of the emission intensity at 665 nm to that at 620 nm, multiplied by 104.

Cholesterol efflux assays
THP-1 cells were used in cholesterol efflux assays to allow for cholesterol-loading of the cells. THP-1 cells were obtained from ATCC (Manassas,VA) and were grown in RPMI medium (Sigma, catalog no. R8005) containing 10% FCS, 0.05 µM 2-mercaptoethanol, 1 mM sodium pyruvate, 2 mM L-glutamine, and antibiotic-antimyotic solution (Sigma, catalog no. A9909, 100 U/ml penicillin, 0.1 µg/ml streptomycin, 0.25 µg/ml amphotericin). THP-1 cells were differentiated into macrophages in 6-well tissue culture dishes at a density of 1 million cells/well by incubation in the same medium plus 100 nM tetradecanoyl phorbol acetate for 3 d. Then the cells were simultaneously labeled with 10 µCi/ml 3H-cholesterol (30) and cholesterol-loaded by incubation for 48 h with 100 µg/ml acetylated low density lipoprotein (LDL). Following this, cells were washed and incubated an additional 24 h in serum-free media containing 1 mg/ml BSA to allow for equilibration of 3H-cholesterol with intracellular cholesterol. Efflux was initiated by adding 10 µg/ml apoAI, with or without compounds, in serum-free medium. After 24 h, media were harvested and cells dissolved in 0.1 M NaOH. Media were briefly centrifuged to remove nonadherent cells and aliquots of both the supernatants and the dissolved cells were subjected to liquid scintillation spectrometry to determine radioactivity. Cholesterol efflux is expressed as a percentage, calculated as [3H-cholesterol in medium/(3H-cholesterol in medium + 3H-cholesterol in cells)] x 100%.

In vivo studies
Normal Sprague Dawley rats (n = 5 per group) were treated for 5 d by gavage with vehicle, 30 mg/kg F3MethylAA, or 30 mg/kg PBFuranAA. The vehicle used in this experiment was 0.5% methylcellulose. Cholestyramine at a concentration of 2% wt/wt was administered in a Harlan Teklad (Indianapolis, IN) diet (LM-485). This diet was reformed into 1/2" pellets as was the control diet. Livers were collected and quick frozen in liquid nitrogen and saved for analysis. RNA was extracted using the Trizol reagent (Life Technologies, Inc.) according to manufacturer’s instructions. Liver cyp7a mRNA levels were measured by Northern blot (see below).

Cyp7a enzyme assays
Rat liver microsomes were prepared as described by Straka et al. (31). Microsomal cyp7a was determined using a variation of the method of Van Cantfort et al. (32). Microsomal protein (1.25 mg/ml) was preincubated at 37 C with 50 µM 3H-cholesterol solubilized in 0.2% Triton X-100 in a buffer containing 100 mM potassium phosphate (pH 7.4), 1 mM EDTA, 50 mM NaF, and 2.5 mM DTT. After 15 min, the reaction was initiated by the addition of a NADPH-regenerating system containing (final concentrations): 20 mM glucose-6-phosphate, 1 mM NADP and 0.75 U/ml glucose 6-phosphate dehydrogenase (Sigma, catalog no. G-8289). Control (blank) reactions were identical except they lacked NADP. The reaction was allowed to proceed for various times (10–45 min) at 37 C and then the reaction was terminated by the addition of 1 ml of chloroform:methanol 1:1 followed by 0.8 ml of water. The extractions were mixed thoroughly, centrifuged briefly to break the phases, and then aliquots of the upper aqueous phase were subjected to liquid scintillation spectrometry to quantify the 3H2O released from the 7-3H-cholesterol. Radioactivity in the blank reactions were subtracted for each time point, and then linear enzymatic reaction rates were determined from graphs of the time courses.

RNA quantitation by real-time PCR
Total RNA was isolated by the use of the Trizol reagent (Life Technologies, Inc.) according to manufacturer’s instructions. This protocol was modified by substituting 1-bromo-3-chloro-propane (Sigma) for chloroform. The RNA was treated with DNase (Ambion, Inc.) before analysis by real-time quantitative RT-PCR to quantify the mRNA levels of target genes (33). Genes and probes are described in Table 1Go. RT and PCR reactions were performed according to PE Applied Biosystems’ protocol for TaqMan Gold RT-PCR and the TaqMan Universal PCR Master Mix. Probes for the genes of interest were labeled with FAM fluorescent dye. The normalizing gene for the human experiments was the human 23-kDa highly basic protein tagged with a VIC dye.


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  		Table 1. RT-PCR probe sets utilized to measure specific gene expression

 
Northern blot analysis
Total RNA was extracted from livers that had been flash-frozen in liquid nitrogen using Ultraspec Reagent (Biotecx, Houston, TX). These RNAs were separated on a 1% agarose (Seakem Gold) in 20 mM [3-N-morpholinopropane sulfonic acid], 8 mM sodium acetate, 1 mM EDTA. RNA was transferred to Duralon membrane by the wicking protocol with 20x SSPE (3.6 M NaCl; 0.2 M NaH2PO4; 0.02 M EDTA, pH 7.7). RNAs were cross-linked to the membrane using a Stratalinker (Stratagene, La Jolla, CA). Blots were prehybridized for 60 min and hybridized overnight at 42 C 50% formamide, 10% dextran sulfate, 1% sodium dodecyl sulfate, 0.9 M NaCl and 0.2 mg/ml low molecular weight salmon sperm DNA. Washing was carried out at 55 C with 0.1x SSPE/0.1% sodium dodecyl sulfate. Bands were detected by exposing the filters to a PhosphorImager screen and imaging with Amersham Biosciences (Piscataway, NJ) Storm and ImageQuant analysis package.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
F3MethylAA binds to LXR {alpha} and ß
As an approach to identify human LXR ligands, a scintillation proximity ligand binding assay (LXRSPA) was developed for LXR{alpha} and LXRß by taking advantage of [3H2]- F3MethylAA, previously identified in a cell based transactivation screen. In LXRSPA, both oxysterols and synthetic ligands (Table 2Go) were characterized (Fig. 1Go). In these experiments F3MethylAA was determined to be a ligand for both LXR{alpha} and LXRß with Ki values of 13 nM and 7 nM, respectively. A naturally occurring ligand, 24(S), 25 epoxycholesterol binds LXR{alpha} with a Ki of 225 nM and LXRß with a Ki of 51 nM. These values are in good agreement with the 200 nM Ki reported for both {alpha} and ß (4). In contrast, 22(R)-OH cholesterol is a much weaker ligand that did not reach a plateau in this assay. PBFuranAA, another synthetic compound that is closely related to F3MethylAA, also binds with a lower affinity.


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  		Table 2. Chemical structures of compounds used

 


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  		Figure 1. F3MethylAA is a ligand for LXR in the SPA. LXRSPA competitive binding experiments were performed to characterize MRL synthetic LXR ligands and compare them with oxysterols. Shown here are dose-response analyses for F3MethylAA 22(R):-OH Cholesterol, 24.(S),25-epoxycholesterol and PBFuranAA on LXR{alpha} (A) and LXRß (B). Data are representative of at least four determinations with SEM.

 
F3MethylAA is a novel LXR agonist
We developed an in vitro cofactor association assay to determine if F3MethylAA functions as an agonist upon binding to the ligand binding domains of LXR{alpha} and LXRß. As depicted in Fig. 2Go, both F3MethylAA and 22(R)-OH cholesterol stimulated the association of recombinant SRC1 coactivator protein with recombinant LXR{alpha} or LXRß ligand binding domains. As expected from LXRSPA, PBFuranAA did not produce an increase in the LXR-SRC1 interaction. These results clearly show that F3MethylAA can interact directly with LXRs in an agonist fashion.



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  		Figure 2. F3MethylAA directly interacts with both LXRs. A cofactor association assay was used to determine if the direct interaction of F3MethylAA with either LXR{alpha} (A) or LXRß (B) results in agonist activity. These data demonstrate the ability of a LXR agonists to induce the association of a fragment of LXR protein with SRC1 polypeptide. The data are reported as the fold increase of the ratio of emission intensity at 665 nm to that at 620 nm. Results are the means of triplicates with SEM.

 
Both F3MethylAA and PBFuranAA are structurally similar, but distinct from 22(R)-OH cholesterol or other steroids (Table 2Go). Both compounds have been previously shown to possess agonist activity against one or more PPAR isoforms (34). On mouse PPAR{gamma} and PPAR{delta} (but not PPAR{alpha}) receptors, these compounds exhibited significant and nearly identical agonist activity with potencies of less than 500 nM. In contrast, using similar human PPAR assays, we previously showed that both compounds are also PPAR{alpha} agonists with potencies of less than 100 nM. Therefore, whereas both F3MethylAA and PBFuranAA have PPAR agonist activity, they differ in that F3MethylAA is a LXR agonist and PBFuranAA is devoid of any LXR activity.

The specificity of F3MethylAA and PBFuranAA for LXRs was determined by screening against a panel of human nuclear receptors. Both compounds were evaluated in binding assays on glucocorticoid receptor, estrogen receptors {alpha} and ß, thyroid receptors {alpha} and ß, and farnesoid X receptor and found not to bind with significant affinities at concentrations up to 10 µM. Additionally, these compounds were tested in a farnesoid X receptor cofactor association assay and also found to be inactive. Furthermore, both compounds were also tested in a Gal4 chimeric transactivation assays for the pregnane X receptor and RXRs and again found to have no activity at concentrations of 10 µM or less. Thus, for experiments described below, PBFuranAA provides an ideal control for F3MethylAA to establish which effects are mediated through LXR.

Cell-based transcription assays using a chimeric LXR constructs were established to measure the relative ability of selected compounds to function as agonists or antagonists for LXR{alpha} and/or LXRß. These assays used fusion proteins with the yeast Gal4 DNA binding domain connected to the hinge region and the LBD of either LXR receptor. Compound titrations were performed using synthetic compounds or selected oxysterols reported in the literature to be LXR agonists (4, 5, 6). In this assay, F3MethylAA was identified as an LXR{alpha} and ß agonist, whereas the structurally related molecule PBFuranAA was inactive (Fig. 3Go). Furthermore, F3MethylAA achieves greater maximal expression of the reporter gene than either 22(R)-OH cholesterol or 24(S),25-epoxycholesterol. In addition, significant activity was detected at lower concentrations than could be observed with either oxysterol ligand. Thus, PBFuranAA was used in several studies as a negative control to elucidate those effects of F3MethylAA that were LXR mediated.



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  		Figure 3. F3MethylAA is more active in a cell-based transactivation assay than selected oxysterols. LXR/Gal4 transactivation assays were performed using a luciferase reporter construct to determine the relative agonist activity of F3Methyl and PBFuranAAs compared with published LXR agonists. Dose-response curves were run with these compounds on both LXR{alpha} (A) and LXRß (B). Each point represents the mean ± SEM of three determinants. The experiment was repeated two or more times with similar results.

 
Previous reports have shown that ligand affinities can vary even between nuclear receptors with high sequence similarity from different species (35). For this reason, we wanted to ensure that the activity seen on the human receptors was similar to that in other species. Therefore, rat and hamster LXRs were cloned into the same Gal4 chimeric construct as was used for the human LXRs. Comparison of the predicted protein sequences from rat, mouse, hamster, and human showed that they differ by only one to three amino acids in the ligand binding domain. F3MethylAA produced similar dose response curves for human, hamster and rat LXR homologs (Fig. 4Go). 22(R)-OH cholesterol and 24(S),25 epoxycholesterol were also assayed on these receptors to determine if their transactivation activity was species dependent; no differences were observed (data not shown). In addition, PBFuranAA was found to be inactive against the rat and hamster LXR homologs (not shown).



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  		Figure 4. F3MethylAA shows no species differences in transactivation assays. Luciferase based transactivation assay were performed using hamster, rat and human Gal4 chimeras. These receptor constructs were exposed to increasing concentrations of F3MethylAA and shown to have similar potencies and activities on the different species homologues of LXR{alpha} (A) and LXRß (B). Each point represents the mean ± SEM of three determinants.

 
Activation of LXR is sufficient for the induction of rat cyp7a
Cyp7a (EC 1.14.13.17) is an important rate-limiting enzyme for production of bile acids from cholesterol. The gene encoding this enzyme, CYP7a, has recently been implicated as an LXR target gene (9). Figure 5Go shows that F3MethylAA is able to induce cyp7a mRNA levels by almost 5-fold over basal activity in primary cultured rat hepatocytes. When cells were incubated with 50 µM chenodeoxycholic acid, there was a suppression of basal cyp7a mRNA levels, as expected (36, 37). The combined treatment of F3MethylAA and chenodeoxycholate showed that there is a significant increase in cyp7a message over basal despite the suppression by chenodeoxycholate. This result further suggests that a LXR agonist is capable of overcoming the negative feedback generated by 50 µM of chenodeoxycholate on the CYP7a promoter in vitro.



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  		Figure 5. F3MethylAA is capable of inducing cyp7a mRNA even in the presence of chenodeoxycholate. Rat primary hepatocytes were treated for 48 h with F3MethylAA in the presence or absence of chenodeoxycholate. Northern blot analysis of cyp7a mRNA is presented here as percent of vehicle. Data have been normalized to glyceraldehyde 3-phosphate dehydrogenase levels. Represented data are the mean of triplicates and their respective standard deviations. All changes are statistically significant when compared with the vehicle control (*, P < 0.05). The combination of F3MethylAA and chenodeoxycholate was also significantly different when compared with chenodeoxycholate treatment alone (**, P < 0.001). There was not a statistical difference between F3MethylAA alone and when administered with chenodeoxycholate.

 
To more clearly establish that LXR activation is sufficient for the induction of cyp7a, the effects of F3MethylAA on cyp7a were characterized in vivo in normal SD rats. Cholestyramine was used as a positive control in the in vivo study as it is known to promote cyp7a induction via a distinct mechanism of disrupting the feedback loop which normally suppresses bile acid production. After 5 d of oral dosing, liver samples were analyzed for both cyp7a mRNA levels and enzyme activity. Treatment with F3MethylAA at 30 mg/kg generated a significant increase in both cyp7a mRNA and enzyme activity (Fig. 6Go). This shows that this ligand is functional not only in cell culture but also in a whole animal model. The results also suggest that a LXR agonist can induce enzyme activity in an intact animal even when the potential induction of bile acid synthesis might be expected to suppress cyp7a promoter activity. Importantly, we also determined that at the same dose PBFuranAA failed to induce cyp7a in rats (Fig. 6Go). Both compounds have been shown to have very similar pharmacokinetic profiles in rats (Leung, K., unpublished results). Therefore, using PBFuranAA as a control, these data indicate that LXR activation by F3MethylAA was sufficient for the in vivo induction of cyp7a in rats.



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  		Figure 6. Rat in vivo studies show that F3MethylAA is able to induce both cyp7a mRNA and enzyme activity. F3MethylAA and PBFuranAA were administered to rats at 30 mg/kg for 5 d followed by analysis of their liver cyp7a mRNA and enzyme levels. Cyp7a mRNA levels were determined by northern analysis and normalized to actin to control for RNA loading. Both cyp7a enzyme and mRNA levels are means and SEM of five replicates and reported as a percent of vehicle (t test values are: *, P = 0.02 and **, P < 0.01). The cyp7a enzyme activity for PBFuranAA was not determined (ND).

 
Activation of LXR does not promote cyp7a induction in human hepatocytes
As shown above, activation of LXR by F3MethylAA was sufficient to induce cyp7a gene expression in rat hepatocytes and in vivo in rat liver. A critical issue involves the extent to which LXR may regulate cyp7a in humans. In studies conducted using primary cultured human hepatocytes, we were surprised to find that relatively high concentrations of F3MethylAA failed to mediate an increase in cyp7a expression (Fig. 7AGo). The same was true for the oxysterol, 22(R)-OH cholesterol and, as expected, with PBFuranAA. In these experiments, compounds were administered for a total of 30 h. A second, fresh application of compound was added 6 h before RNA was prepared to allow for the analysis of both acute and chronic treatment effects. To be sure that the compounds were cell penetrant and active, we took advantage of the fact that both F3MethylAA and PBFuranAA are agonists for human PPAR{alpha}. Thus, both compounds produced significant increases in the expression of a well characterized PPAR{alpha} target gene, carnitine palmitoyl transferase I (38, 39) (Fig. 7AGo). Additionally, in a second human primary hepatocyte preparation, F3MethylAA induced sterol response element binding protein 1 (SREBP1), stearoyl-coenzyme A desaturase (SCD), and the gene encoding ATP binding cassette protein G1 (ABCG1) by 3.5-, 6.8-, and 5.3-fold, respectively. Induction of the same genes by 22(R)-OH cholesterol were 4.9-, 3.6-, and 5.6-fold. Again, neither treatment induced cyp7a mRNA expression and in fact, slightly suppressed its levels. The discrepancy between the effects of LXR activation on cyp7a in rat and human liver cells suggests the potential for a species difference in the cyp7a promoter.



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  		Figure 7. In human primary hepatocytes and macrophages, F3MethylAA induced mRNA levels of ABCA1 but not cyp7a. Human primary hepatocytes (7A) or macrophages (7B) were treated with 5 µM F3MethylAA or PBFuranAA or 15 µM 22(R)-OH cholesterol for a total of 30 h, and their effects on specific genes were determined by RT-PCR. RNA was normalized to the internal control of 23-kDa highly basic protein and expressed as fold change over the vehicle control. Values are the average of two determinations. Similar results were seen in at least two additional experiments.

 
Activation of LXR target genes in human macrophages
Because cyp7a in liver was not affected by LXR activation, we examined the expression of other potential LXR responsive genes in human macrophages. ABCA1 mRNA levels were substantially increased by F3MethylAA and 22(R)-OH cholesterol but not by PBFuranAA (Fig. 7BGo). Additionally, LXR activation by F3MethylAA (but not PBFuranAA) was sufficient to elevate the steady-state mRNA levels of two other LXR target genes, ABCG1 and SREBP1 (22, 25, 40, 41, 42) (Fig. 7BGo). These results indicate that F3MethylAA is an agonist of endogenous human LXR. Furthermore, the results of these studies strongly support the hypothesis that ABCA1, ABCG1 and SREBP are bona fide LXR target genes in humans but that cyp7a is not. This implies that while rats and mice respond to LXR agonism with cyp7a elevation, humans do not regulate cyp7a via a LXR-mediated pathway.

Activation of LXR stimulates cellular cholesterol efflux onto apoAI
The recent discovery that Tangier disease is caused by mutations in the ABCA1 gene demonstrates a connection between ABCA1 message levels and the ability of key tissues to export cholesterol and to maintain HDL levels (18, 19, 20, 21). Although the exact mechanism by which ABCA1 mediates cellular sterol efflux has not been elucidated, there appears to be a requirement for apoAI as an acceptor (24, 43, 44). To assess whether LXR activation would promote cellular cholesterol efflux, we studied the effect of F3MethylAA on efflux of 3H-cholesterol from the human THP-1 cells. As depicted in Fig. 8AGo, F3MethylAA produced a dose-dependent increase in cholesterol efflux within the sub-micromolar range of concentrations with which it was shown to activate human LXR. In contrast, PBFuranAA did not induce cellular cholesterol efflux from these cells. In THP-1 macrophages a significant increase in efflux was apparent in both normal and cholesterol-loaded cells when both apoAI and F3MethylAA were present (Fig. 8BGo). Cholesterol loading itself induced a slight increase in cholesterol efflux from cells relative to unloaded cells.



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  		Figure 8. F3MethylAA was able to induce cholesterol efflux in THP-1. A cholesterol efflux assay was performed in the human THP1 cells. A, THP1 cells were treated with a dose-response curve of F3MethylAA and the amount of effluxed 3H-cholesterol was measured in the media. B, Either normal or cholesterol-loaded THP-1 macrophages were assayed for their ability to efflux cholesterol in response to treatment with F3MethylAA. For both figures data are the average of triplicates and SEM and reported as the percent of the vehicle control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The LXRs are members of the nuclear receptor family and manifest their function by regulating the transcription of specific target genes. LXR{alpha} has been implicated in the regulation of cholesterol homeostasis based principally on the inability of LXR{alpha} knockout mice to cope with the challenge of a high cholesterol diet (9). Importantly, cholesterol feeding in wild-type mice induced the expression of cyp7a, whereas LXR{alpha} knockout mice were incapable of recognizing elevated cholesterol levels and failed to induce cyp7a transcription. Thus, they could not increase cholesterol clearance via its conversion to bile acids. Knockout mice have been an important tool in elucidating the function of proteins. Another approach to determine the function of a receptor is to characterize the biological effects of potent synthetic ligands. Toward this end, we describe here the characterization of a novel LXR agonist.

Since the identification of cyp7a as a potential LXR target gene, ABCA1, was suggested to be regulated by LXR agonism (17, 22, 23, 24). ABCA1 is involved in the return of cholesterol from the periphery to the liver in a process that is referred to as reverse cholesterol transport. This suggests a coordinate pathway whereby excess cholesterol is sensed by LXR(s) in peripheral cells, inducing ABCA1 levels and initiating reverse cholesterol transport. Subsequent LXR-mediated hepatic cholesterol 7{alpha}-hydroxylase induction could allow for the elimination of excess cholesterol from the body.

F3MethylAA was characterized as a novel and structurally distinct LXR agonist. The LXR activity was similar for both {alpha} and ß and in several species including both human and rat, allowing for experiments to be conducted in both species. In addition, use of a cell-free fluorescent coactivator association assay for LXR agonism established that F3MethylAA was active as a direct LXR ligand and agonist. Although F3MethylAA also has PPAR activity, it was not active in several other nuclear receptor assays. Moreover, we used a closely related analog of F3MethylAA, PBFuranAA, which was devoid of LXR activity but possessed nearly identical PPAR activity and rat pharmacokinetic properties as a negative control.

The use of F3MethylAA and PBFuranAA clearly established that LXR activation was sufficient to promote cyp7a induction in rat liver cells in vitro and in vivo. Our data in rats extend results previously reported (25) for mice using a distinct synthetic LXR ligand, T-0901317. Mice treated for 7 d with T0901317 showed a 3-fold induction of cyp7a mRNA in the liver. T0901317 also mediated induction of ABCA1, SREBP1 and SCD mRNA in mice and SREBP1c in McA-RH7777, a rat liver cell line (17, 25, 45). The results presented by these authors are also shown to be LXR mediated in that they are abolished in the LXR double knockout mice. Our data extend these results to human primary hepatocytes, with the notable exception of cyp7a.

Thus, our data conclusively demonstrate that that the induction of the endogenous cyp7a gene by LXR agonism is species dependent. Although F3MethylAA was able to induce cyp7a gene expression in rats, F3MethylAA did not alter cyp7a mRNA levels in human primary hepatocytes. The lack of effect on cyp7a cannot be ascribed to a failure of F3MethylAA to enter the cells because other LXR-responsive genes were induced by F3MethylAA in primary human hepatocytes. Furthermore, the well studied oxysterol LXR agonist 22(R)-OH cholesterol also induced ABCA1 but failed to regulate cyp7a in human primary hepatocytes. Our data indicate that cyp7a is controlled by LXR in a species-dependent manner.

Species differences for induction of cyp7a have been previously suggested. Although mice and rats respond to cholesterol feeding with induction of cyp7a, neither hamsters (12) nor rabbits (13) show this response. Humans also appear to lack this response (14). This species difference is also supported by cyp7a promoter analysis (15). In EMSAs, Chiang et al. (15) showed that the LXRE from mouse, rat, and hamster cyp7a promoter was shifted in the presence of LXR{alpha} and RXR. Conversely, the LXRE from the human cyp7a promoter was not shifted. Cyp7a promoters driving luc from these species also showed the same pattern of responsiveness. It is of interest to note that species that fail to induce cyp7a upon cholesterol-feeding (hamsters, rabbits, humans) all express cholesterol ester transfer protein (CETP) (46), which is an LXR-responsive gene (47). Conversely, those species that increase cyp7a upon cholesterol feeding do not have CETP. Unlike cyp7a, ABCA1 appears to be controlled by LXR in all species studied to date. These observations suggest that ABCA1-mediated cholesterol efflux from peripheral tissues onto HDL is a general phenomenon, whereas subsequent handling of the HDL cholesterol differs between species. In some mammals (rats and mice), the HDL cholesterol is taken up by the liver where LXR action increases cyp7a-catalyzed hydroxylation of excess cholesterol. In other mammals, such as hamsters, rabbits, and humans, LXR action induces expression of CETP, which enhances the transfer of cholesteryl ester from HDL to LDL. The elevated LDL cholesterol caused by cholesterol feeding in these species could be viewed as a storage form of cholesterol.

Using F3MethylAA, we were able to provide new evidence that ABCA1 is an LXR-responsive gene in human cells. Furthermore, given the induction of ABCA1 by LXR agonists, we were also prompted to investigate whether LXR activation could be sufficient to increase efflux of cholesterol from a relevant human cell type. Thus, using THP1 monocyte-macrophages, we established that F3MethylAA could produce cellular cholesterol efflux in an apoAI-dependent fashion. These results provide an independent line of evidence which bolsters the hypothesis that LXR is a major regulator of ABCA1 gene expression and reverse cholesterol transport. Also, a second ABC family member, ABCG1 (ABC8) was induced in human hepatocytes by F3MethylAA. This is in agreement with a published report that ABCG1 is LXR responsive in mice (22).

Taken together, our results confirm that LXR agonism leads to the induction of genes that are important in cholesterol transport and catabolism was well as those that are involved in fatty acid synthesis. ABCA1 is now well documented to be involved in the movement of cholesterol out of the cell and onto apoAI. ABCG1 has also been suggested to play a role in cholesterol transport (48). Additionally, our work with this LXR agonist and human primary cells supports the observation of Repa et al. (40) and Schultz et al. (25) that in rodents and humans, LXR agonism will lead to the increase of SREBP1 and the resultant increase of lipogenic genes such as fatty acid synthase and SCD as well as predict the mechanism whereby LXR agonism will lead to elevated triglyceride levels.

In summary, our studies with the novel LXR ligand F3MethylAA confirm that ABCA1, ABCG1, and SREBP1 are responsive to LXR agonism. Importantly, we also show that cyp7a regulation by LXR agonism is species specific. As reported (9), LXR agonism will mediate the induction of cyp7a mRNA in mice. However, our studies in human primary hepatocytes show that even though ABCA1, ABCG1 and SREBP1 are induced by exposure to LXR agonists (F3MethylAA and 22(R)-OH cholesterol), these ligands fail to modulate cyp7a mRNA levels. These studies demonstrate that work with synthetic compounds, such as F3MethylAA, mimic the biological effects seen in vitro with an oxysterol agonist, such as 22(R)-OH cholesterol. These results also demonstrate the usefulness of experiments with synthetic ligands to elucidate the function of the LXRs.


   Acknowledgments
 
We thank John Capone for supplying the LXR{alpha} cDNA. We also thank Cristopher Sondey, Julia Ayala, Joel Yudkovitz, Jisong Cui, Kwan Leung, and Elizabeth Birzen for human primary monocyte isolation, compound synthesis, additional receptor counterscreens, and pharmacokinetic analysis. We also acknowledge Georgianna Harris for the critical review of this manuscript.


   Footnotes
 
1 Current address: University of Pennsylvania Medical School, MicroArray Core Facility, 277 John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6082. Back

Abbreviations: ABCA1, Gene encoding ATP binding cassette protein A1; ABCG1, gene encoding ATP binding cassette protein G1; apoAI, apolipoprotein AI; CETP, cholesterol ester transfer protein; CS-FCS, charcoal-stripped fetal calf serum; CYP7a, cholesterol 7{alpha}-hydroxylase; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; F3MethylAA, 3-chloro-4-(3-(7-propyl-3-trifluoromethyl-6-(4,5)-isoxazolyl)propylthio)-phenyl acetic acid; GST, glutathione S-transferase; HDL, high density lipoprotein; Ki, equilibrium dissociation constant; LBD, ligand binding domain; LDL, low density lipoprotein; luc, luciferase; LXR, liver X receptor; LXRE, LXR response element; LXRSPA, LXR scintillation proximity assays; PBFuranAA, (3-chloro-4-(3-(3-pheny-7-propylbenzofuran-6-yloxy)propylthio)-phenyl acetic acid); PPAR, peroxisome proliferator-activated receptor; RPMI, Roswell Park Memorial Institute; RXR, retinoid X receptor; SCD, stearoyl-coenzyme A desaturase; SRC-1, steroid receptor coactivator; SREBP1, sterol response element binding protein 1.

Received November 6, 2001.

Accepted for publication March 20, 2002.


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 Introduction
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 Results
 Discussion
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