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Molecular Endocrinology, doi:10.1210/me.2003-0477
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Molecular Endocrinology 18 (8): 2000-2010
Copyright © 2004 by The Endocrine Society

Liver X Receptors (LXRs) Regulate Apolipoprotein AIV-Implications of the Antiatherosclerotic Effect of LXR Agonists

Yu Liang, Xian-Cheng Jiang, Ruijie Liu, Guosheng Liang, Thomas P. Beyer, Hong Gao, Timothy P. Ryan, Shuyu Dan Li, Patrick I. Eacho and Guoqing Cao

Lilly Research Laboratories (Y.L., T.P.B., H.G., T.P.R., S.D.L., P.I.E., G.C.), Eli Lilly & Company, Indianapolis, Indiana 46285; Department of Anatomy and Cell Biology (X.-C.J., R.L.), SUNY Downstate Medical Center, Brooklyn, New York 11203; and Department of Molecular Genetics (G.L.), University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046

Address all correspondence and requests for reprints to: Guoqing Cao, Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, Indiana 46285. E-mail: Guoqing_Cao{at}lilly.com.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Liver X receptors (LXRs) regulate target genes that are critical in lipoprotein metabolism and atherosclerosis. Apolipoprotein AIV (ApoAIV) is an apolipoprotein that is associated with chylomicrons and high-density lipoproteins. Plasma ApoAIV level in humans is inversely correlated with coronary artery events and overexpression of ApoAIV in mice results in significant reduction in atherosclerosis. We report here that LXRs directly regulate apoAIV at the transcriptional level. Treatment of C57B6 mice with a synthetic LXR agonist, T0901317, resulted in significant increases in plasma apoAIV that was associated with high-density lipoprotein. Examination of both intestinal and liver apoAIV mRNA revealed specific increases in liver mRNA only. In a human heptoma HepG2 cell model, apoAIV mRNA was up-regulated upon the treatment with either native or synthetic LXR agonists. Nuclear run-on study revealed a significant increase in the ApoAIV transcriptional rate upon LXR activation. Examination of the human apoAIV proximal promoter revealed a potential LXR response element that demonstrated binding with HepG2 nuclear extracts. Cotransfection studies in HepG2 cells indicated that this responsive element was functional in mediating the human ApoAIV gene response to LXR agonists. In addition, we identified a functional LXR-responsive element at 3' end enhancer region of mouse ApoAIV gene. We conclude that ApoAIV is a direct target gene of LXRs that may contribute to the antiatherogenic effect of LXR activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LIVER X RECEPTORS (LXRS) are oxysterol receptors that mediate cholesterol homeostasis (1). LXRs regulate ATP binding cassette transporter A1 (ABCA1) (2, 3), which is essential and rate-limiting in mediating cellular cholesterol efflux (4). LXRs also regulate apolipoproteins (ApoE and ApoC), which act as cholesterol acceptors in the cholesterol efflux process (5, 6, 7). High-density lipoprotein (HDL) modifying lipid transfer proteins including cholesterol ester transfer protein and phospholipid transfer protein are also LXR target genes (8, 9). Furthermore, LXRs regulate ABCG5 and ABCG8 in the liver and in intestines resulting in increased cholesterol excretion into the bile and decreased cholesterol absorption, respectively (10, 11). Thus, LXRs are regarded as master transcription factors mediating cholesterol catabolism. In addition, LXRs regulate other metabolic pathways including lipogenesis (12, 13), gluconeogenesis (14), and inflammation (15).

LXRs are ligand-activated nuclear receptors (16). Several oxysterols have been identified as native LXR ligands (17, 18, 19). Synthetic LXR agonists have been developed, and the specificity in regulating LXR target genes was demonstrated in mice deficient in LXR expression (12, 20). LXRs function with retinoid X receptor (RXR) as an obligatory permissive heterdimer and regulate gene expression through the hormone-responsive element separated by four nucleotides (DR-4) that is located in the enhancer regions of target genes (16). Administration of synthetic LXR agonists in mice led to increased HDL cholesterol and HDL particle size along with triglyceride accumulation in the liver (8, 12). The anticipated antiatherosclerosis property of LXR activity has been demonstrated in rodent models (21, 22, 23).

Apolipoprotein AIV (ApoAIV) is an apolipoprotein that is primarily synthesized in intestines and to a lesser extent in the liver (24). In humans, it is associated with chylomicrones and is thus believed to play a role in lipid absorption and lipoprotein assembly in intestines (25). In fasting human plasma, apoAIV is either associated with HDL particles (26) in which it can activate lecithin-cholesterol acyltransfer protein (27) or exists in free form in lymph and plasma (28), where it may play critical roles in mediating cholesterol efflux (7, 29, 30) and provide protection against oxidant insult to vessel wall (31, 32). These data are consistent with the observation that apoAIV is antiatherogenic as demonstrated by studies in transgenic mice (29, 32, 33). Human genetic studies also demonstrated inverse correlation of plasma apoAIV level with coronary events (34, 35). Recently, several reports demonstrated apoAIV expression in brain that may modulate feeding behavior in rodents (36, 37).

In the present studies, we have observed that apoAIV is regulated by LXRs. Ligand activation of LXRs led to an increased expression of apoAIV at the transcriptional level. Further analysis revealed that apoAIV was a direct target gene of LXRs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previously, we have observed that an LXR agonist, T0901317, increased HDL cholesterol and HDL particle size in C57B6 mice (8). Fasting plasma samples from C57B6 mice treated with vehicle or T0901317 at 10 or 50 mg/kg were subject to fast protein liquid chromatography (FPLC) and the isolated lipoproteins were pooled (1:20–23, 2:24–27, 3:28–31, 4:32–36, and 5:37–41; also see Fig. 1AGo) for apoAIV Western blot analysis. In vehicle-treated animals, apoAIV was largely associated with HDL particles (Fig. 1BGo). T0901317 treatment resulted in an increase in HDL levels (fraction nos. 4 and 5) and also in HDL particles of increased size (fraction no. 3) (8). Treatment of mice with T0901317 resulted in an apparent increase in plasma apoAIV levels that were primarily associated with HDL cholesterol, along with elevation in the enlarged HDL fractions (Fig. 1BGo).



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  		Fig. 1. Increased Plasma apoAIV Level upon LXR Activation

C57B6 mice were treated with either vehicle or daily doses (10 and 50 mg/kg) of T0901317 for 7 d. Plasma samples were fractionated with FPLC (A) and pooled samples (labeled as 1–5) were analyzed with Western blot analysis with an apoAIV polyclonal antibody (B). Fraction three represented the HDL of increased particle size, and fractions four and five represented HDL particles.

 
ApoAIV is primarily synthesized in intestines and to a much lesser extent in the liver in C57B6 mice (38). We examined mRNA levels in both liver and intestines in animals with or without T0901317 treatment. In the liver, the basal apoAIV mRNA level was much lower than that of intestine, but T0901317 treatment led to a dramatic increase in apoAIV mRNA levels in a dose-dependent fashion (Fig. 2Go). Intestinal ApoAIV mRNA was largely expressed within duodenum and jejunum. No change in ApoAIVA mRNA levels were observed with T0901317 treatment in intestines (Fig. 2Go). Thus, a specific synthetic LXR agonist up-regulated apoAIV mRNA in the liver specifically and consequently increased plasma apoAIV levels that was primarily associated with HDL.



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  		Fig. 2. LXR Agonist T0901317 Regulates ApoAIV Expression in Mouse Liver in Vivo

ApoAIV mRNA expression levels were analyzed by Northern blot on RNA samples from mouse liver and intestine with or without T0901317 treatment. Equal amounts of total RNA were loaded onto each lane of agarose-MOPS-formaldehyde gels, after which the Northern blot analysis was performed as described in Materials and Methods. Tubulin was used as a loading control.

 
To explore whether similar regulation of apoAIV also exists in humans, we used a heptoma cell line, HepG2, of human origin for further analysis. HepG2 cells were treated with a native ligand of LXR [22-(R)-hydroxycholesterol], a ligand of RXR [9-cis-retinoic acid (RA)], a synthetic LXR agonist (T0901317), an oxysterol that has no LXR agonist activity [22-(S)-hydroxycholesterol] or combined treatment of both LXR agonist and RXR agonist. Quantitative real-time PCR analysis was used to quantify apoAIV mRNA levels. As shown in Fig. 3AGo, both LXR agonists and the RXR agonist can effectively regulate apoAIV mRNA levels. The synthetic agonist T0901317 appeared to be more efficacious than native ligands in inducing apoAIV mRNA. 22-(S)-hydroxycholesterol had no activity in up-regulating apoAIV mRNA suggesting an LXR basis for the above-mentioned observation. Combined treatment of an LXR agonist along with a RXR agonist appeared to be additive, which was in agreement with the concept that LXR/RXR functions as a permissive heterodimer. To confirm that the increased apoAIV mRNA levels were a result of increased transcription, we conducted nuclear run-on experiments in HepG2 cells. Consistent with the quantitative PCR measurements, increased apoAIV transcription was observed with either LXR or RXR ligand treatment, suggesting transcriptional regulation of apoAIV mRNA in HepG2 cells (Fig. 3BGo). We also examined this potential regulation in Caco2 cells-an intestinal cell model of human origin. We did not find any significant change in apoAIV mRNA levels as measured by Taqman analysis in Caco2 cells upon LXR agonist treatment (data not shown), suggesting a paralleled tissue-specific regulation in both humans and mice.



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  		Fig. 3. LXR and RXR Ligands Induce apoAIV Expression Transcriptionally in HepG2 Cells

HepG2 cells were treated with various LXR/RXR ligands shown in the figure for 24 h. A, RNA was extracted and Taqman assays were performed as described in Materials and Methods. Please note that 22-(S)-hydroxycholesterol, an oxysterol with no LXR agonist activity, did not increase ApoAIV expression. B, Nuclear run-on studies were done in HepG2 cells as described in Materials and Methods. Please note the correlation of nuclear run-on results with the real-time PCR studies on the apoAIV mRNA level. DMSO, Dimethylsulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
We examined the proximal promoter region of human apoAIV gene and found a potential LXR-responsive element (LXRE: DR-4 of hormone-responsive element AGGTCA) at –140 to –124 (Fig. 4AGo). A gel mobility shift assay was employed to examine the interaction of this potential LXRE with HepG2 nuclear extracts (Fig. 4BGo). A shifted band was only present when radiolabeled LXRE was incubated with HepG2 nuclear extracts (lane 2). This band was absent when a mutant LXRE was substituted for wild-type LXRE (lane 9). The specificity of the binding was further confirmed when the binding was effectively competed with 50x cold oligoes of the potential LXRE (lanes 3 and 4) or a standard LXR-responsive element (lanes 5 and 6). The mutant form of the potential LXRE, however, was ineffective in competition (lanes 7 and 8). The formation of the LXR/RXR/LXRE complex was further substantiated by the fact that the addition of either LXR or RXR antibody effectively abolished the LXR/RXR heterodimer interaction with the LXRE (Fig. 4CGo). To explore whether the potential LXRE is functional, we subcloned a 600-bp promoter fragment that contained the potential LXRE (–140 to –124) into a basic firefly luciferase reporter vector. Cotransfection into HepG2 cells of this reporter construct with LXR/RXR expression vectors followed by treatment with LXR or RXR ligands revealed that the promoter reporter construct was indeed responsive to LXR/RXR agonist treatment (Fig. 4DGo). The increase in the activity of the promoter reporter construct was abolished when the corresponding LXRE in apoAIV promoter was mutated, therefore, the LXRE in the proximal promoter of the human apoAIV gene is functional.



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  		Fig. 4. Identification of a Functional LXRE in the Proximal Promoter of Human ApoAIV Gene

A, Sequence alignment of proximal promoters of human and mouse ApoAIV gene. The potential LXRE (DR-4) was underlined and boldface. B, EMSA for the potential LXRE in the proximal promoter of human ApoAIV gene. The oligonucleotides were labeled and the EMSA was performed as described in Materials and Methods. C, Specificity of LXR/RXR/LXRE complex. EMSA was performed as described in Materials and Methods. Five micrograms each of either LXR or RXR antibodies were included in the incubation. D, Cotransfection studies with human ApoAIV promoter-reporter construct and LXR/RXR expression vectors. A human ApoAIV promoter fragment was subcloned into a firefly luciferase reporter vector and transfected into HepG2 cells with LXR and RXR expression vectors under cytomegalovirus promoter. Cells were treated with various LXR/RXR ligands for 24 h, and the luciferase activity was measured as described in Materials and Methods. pGL-2 basic was used as a control.

 
The equivalent position of mouse apoAIV gene contained a potential LXRE with one nucleotide difference compared with the LXRE in the human apoAIV gene promoter (Fig. 4AGo). However, gel mobility shift assay with HepG2 cell nuclear extract did not reveal any significant binding with the mouse form of this potential LXRE in the proximal promoter (Fig. 5BGo, lane 3) suggesting a different regulatory mechanism for mouse apoAIV gene. We examined further the promoter, intron one, and the 3' end sequence within the mouse apoAIV gene and found four potential LXREs near the 5 kilobase 3' end of the mouse apoAIV gene (Fig. 5AGo). Gel mobility shift assay, however, identified only one of these response elements (LXRE03) that showed strong binding to HepG2 cell nuclear extract (Fig. 5BGo, lane 6). Further competition studies with either cold wild-type or the mutant form of the mouse 3' end LXRE03 revealed specificity in binding (Fig. 5CGo). LXR or RXR antibodies effectively abolished the binding suggesting the involvement of LXR/RXR heterodimer formation in this interaction (Fig. 5CGo, lanes 7 and 8). To further confirm the binding nature of the trans-factors with cis elements, we examined the interaction through utilizing the in vitro-translated LXR/RXR recombinant proteins. Similar findings were observed to what were described using HepG2 cell nuclear extracts, confirming the formation of LXR/RXR/LXRE complex (Fig. 5DGo). To study the potential functionality of the responsive element, we subcloned the 1.5-kb fragment containing all the potential responsive elements (Fig. 5AGo) into thymidine kinase (TK)-luciferase context. Cotransfection study, however, revealed that this reporter construct exhibited only 30% increase in reporter activity when LXR agonist was present compared with the control (data not shown) implicating that the cis element may have to function in a specific enhancer context. As an alternative approach, two copies of the LXRE03 were subcloned into a minimum TK promoter-reporter vector and the resulting construct was examined by cotransfection into HepG2 cells with LXR/RXR expression vectors. Treatment of HepG2 cells with either T0901317 or 9-cis-RA resulted in dramatic increases in luciferase reporter activity, suggesting that the 3' end LXRE was potentially a functional response element (Fig. 5EGo). The mutant form of the LXRE03, on the other hand, did not show any significant changes upon ligand treatment (Fig. 5EGo). Examination of the 3' end sequence of human apoAIV gene revealed a potential LXRE at about the equivalent position to that of mouse genome (see Table 1Go for detailed sequence). Although gel mobility shift assay indicated binding to HepG2 nuclear extract (data not shown), transfection studies did not reveal any response to LXR or RXR agonist treatment (Fig. 5EGo), implicating that the potential LXRE at the 3' end of the human ApoAIV gene was not functional.



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  		Fig. 5. A Functional LXRE at the 3' End of Mouse ApoAIV Gene

A, 3' End sequence (5000–6075) of mouse ApoAIV gene (the last nucleotide of the stop codon in the coding region was arbitrarily labeled as 0). Potential LXREs were underlined. B, Specific binding activity of mouseAIV3'-LXRE-03 by HepG2 nuclear extract as assessed by electrophoretic gel mobility shift assay. Oligoes were labeled and the assay was carried out as described in Materials and Methods. The LXRE identified within human ApoAIV proximal promoter was used as a positive control. Only mouse 3' end LXRE03 displayed strong binding activity. C, Specificity of mouse 3' end LXRE03 binding to HepG2 nuclear extract. EMSA and the competition were performed similarly as described in Fig. 4Go, B and C. D, The LXREs in the promoter of human ApoAIV gene and at the 3' end of mouse ApoAIV gene bind to LXR/RXR heterodimer. EMSAs were performed as described in Materials and Methods. Control LXRE, HuAIV-5' LXRE, MutHuAIV-5' LXRE, mAIV-3' LXRE-03, and Mut mAIV-3' LXRE-03 (as shown in Table 1Go) were synthesized with NheI overhangs. LXR and RXR proteins were generated from expression vectors using a coupled in vitro transcription/translation system (Promega). The 32P-labeled probes were incubated with in vitro synthesized LXR and RXR proteins as indicated. Unprogrammed lysates were used as controls (lanes 1, 5, 9, 13, and 17). The specific LXR/RXR binding (lanes 4, 8, and 16) is indicated by an arrow. The nonspecific bands in lanes 13 and 17 (probes bind to unprogrammed lysates) are indicated by an asterisk. E, Cotransfection analysis of the potential LXREs. Two copies of the respective LXREs were subcloned into a minimal TK promoter luciferase vector. Mutant construct were generated in a similar fashion (see the Materials and Methods). Cotransfection into HepG2 cells and luciferase assay were performed similarly as described in Fig. 4DGo. 3xLXRE was used as a positive control, whereas TK-luciferase vector was served as a negative control.

 

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  		Table 1. Oligonucleotides Used in EMSA

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Recent studies have identified LXRs as oxysterol receptors that sense cholesterol levels and mediate cholesterol homeostasis. LXRs regulate a myriad of genes involved in cholesterol balance, including apolipoproteins (ApoE and ApoC) in macrophages. We report here apoAIV as the first apolipoprotein directly regulated by LXRs in the liver.

The physiological role of apoAIV has not been clearly defined. Recent studies have suggested that apoAIV can activate lecithin-cholesterol acyltransfer protein (27), possess antioxidant properties (31, 32), and mediate cholesterol efflux (7, 29, 30). Thus, its potential function is very much similar to that of apoAI except that it is only a minor apolipoprotein component of HDL particles. It is possible that the absence of increased lesion development in apoAI-deficient mice compared with wild-type controls is a result of similar functions that apoAIV mediates. These data are consistent with the observation that apoAIV transgenic mice are antiatherogenic (29, 32, 33). Furthermore, in humans, recent data suggest that like apoAI, apoAIV is inversely associated with coronary artery events (34, 35). LXRs were postulated as antiatherogenic genes based on the fact that they up-regulate ABCA1 (2), apoE (5), and hence the cholesterol efflux process (39). The hypothesis was proven recently with bone marrow transplantation study and two other independent studies with synthetic LXR agonists (21, 22, 23). Our observation suggests that increased plasma apoAIV may contribute significantly to the antiatherogenic effect of LXRs in addition to ABCA1 and apoE regulation.

Unlike some LXR target genes that display species difference (40, 41), we have observed apoAIV regulation by LXRs in both human cells and in mice in vivo. The underlying mechanism responsible for the observed regulation, however, could be very different after careful examination. The human ApoAIV gene resides in the ApoAI-CIII-AIV-AV gene cluster, with multiple potential important cis-acting elements mediating nuclear receptor regulation (42). Here we have identified a functional responsive element in the proximal promoter of the human apoAIV gene. The mouse apoAIV gene, however, lacks the response element at the equivalent position in the proximal promoter and appears to use a completely different mechanism to mediate LXR response. A potential functional responsive element was identified at about 5 kb 3' end of the apoIV gene. Whether this is the precise mechanism through which LXR agonists mediate apoAIV regulation in mice requires further studies. Our studies identified potential responsive elements at 5' promoter and 3' enhancer regions of human and mouse apoAIV gene mediating LXR/RXR heterodimer regulation respectively but in no way exclude the possibility that other mechanisms including additional cis elements that may also contribute to this process. Previously, a pair of LXREs were identified at the 3' end enhancer region of human apoE gene mediating apoE response to LXR agonists (5). Our study provides an additional example that responsive element at 3' end enhancer region may effectively mediate gene regulation for nuclear receptors. Our study also provides an interesting example of differential mechanisms mediating similar responses to the same nuclear receptor between humans and rodents.

Some LXR target genes are regulated in a tissue-specific fashion, implicating the involvement of tissue-specific cofactors mediating transcriptional gene responses in promoter context-specific fashion (8, 43). We have observed dramatic transcriptional regulation of apoAIV in mouse liver upon LXR ligand treatment, whereas intestinal regulation was absent. This could be possibly attributed to the high basal level expression of AIV in intestines (duodenum and jejunum) or the presence of tissue-specific cofactors. Other potential tissues that would mediate LXR regulation of apoAIV are macrophages and the brain. We have not detected apoAIV mRNA signal by Northern blot analysis in mouse peritoneal macrophages with or without LXR agonist treatment. At this moment, we do not know whether this was because of the lack of expression of apoAIV or a lack of LXR regulation in macrophages.

ApoAIV is responsive to high-lipid diet feeding in rodents (38, 44). Several gene products that are feed-forwardly regulated by cholesterol feeding have been identified to be mediated through LXRs (5, 10, 45, 46). Whether LXRs mediate ApoAIV response to dietary feeding remains to be determined.

It has been recently reported that apoAIV is expressed in hypothalamus in the brain locally and may mediate feeding behavior in rodents (36, 47). It will be of great interest to investigate whether apoAIV is regulated by LXRs in the brain. Although T0901317 does not appear to have effects on the feeding behavior in wild-type C57B6 mice (8), it is possible that this effect is either species specific or masked by some compensatory mechanism in wild-type mice. In ZDF rat or Fa/Fa rat, dramatic reduction in food intake was observed even when the compound was used at very low doses that were not sufficient to induce any liver toxicity (14). It is possible that LXR agonists can penetrate blood brain barrier to regulate apoAIV and contribute to the inhibition of food intake. Studies are ongoing to further understand these observations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents and Material
Culture media were purchased from Invitrogen Life Technologies (Carlsbad, CA). 9-cis-RA, 22-(R)-hydroxycholesterol, and 22-(S)-hydroxycholesterol were purchased from Sigma (St. Louis, MO).

Animals
Eight-week-old male C57BL6 mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and acclimated for 2 wk before the experiments. T0901317 was prepared in wet granules (212.5 mg Povidone, 3.77 g Lactose Anhydrous (granular), and 64.8 µl Polysorbate 80 (Tween 80) in 250 ml water). Animals (six per group) were treated with either vehicle or various doses of T0901317 for 7 d and fasted for 15 h before being killed with CO2 euthanasia. Plasma and tissues were then collected and used 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.

Cell Culture
The human hepatoma cell line HepG2 was purchased from ATCC. Cells were maintained in DMEM/F12 (3:1) with 10% FBS. At 80–90% confluency, cells were treated with various reagents for 24 h. At the end of the treatment, total cellular RNA was extracted for real-time Taqman analysis. Cells were also used for transfection with various luciferase constructs for luciferase assay.

Lipoprotein Analysis by FPLC
Lipoprotein analysis was performed as described previously (8). Briefly, plasma samples from animals were prepared and pooled. Two hundred microliters of pooled sample was applied to two Superose 6 size exclusion columns and eluted with PBS (pH 7.4). Cholesterol content of different fractions was measured by a commercial kit (Wako, Richmond, VA).

Western Blot Analysis
Plasma samples were separated by FPLC. Different fractions were pooled (fractions 1:20–23, 2:24–27, 3:28–31, 4:32–36, and 5:37–41) for apoAIV analysis. Designated FPLC fractions were separated on Tris-glycine gels (Bio-Rad, Hercules, CA) under denaturing conditions. Protein was transferred to nitrocellulose membrane and then blotted with a polyclonal antibody to apolipoprotein AIV (a gift from Dr. Charlie Bisgaier, Esperion Therapeutics, Inc., Ann Arbor, MI). Blots were developed with ECL Western blotting detection reagents (Amersham, Piscataway, NJ) and documented using X-OMAT film (Kodak, Rochester, NY).

Northern Blot Analysis
Total cellular RNA was isolated from mouse liver and intestines by using TriZol reagent (Invitrogen Life Technologies). RNA was separated by 1% agarose-MOPS-formaldehyde gel electrophoresis and transferred to nylon membranes. RNA was then hybridized with various [32P]-labeled cDNA probes in ExpressHyb (CLONTECH, Palo Alto, CA). The results were visualized by x-ray autoradiography. For detecting {alpha}-tubulin mRNAs, full-length human cDNA was used. For mouse ApoAIV mRNA blotting, PCR amplified fragments were used (PCR oligonucleotide primers: 5'-CGTATGCTGATGGGGTGCACAA, and 5'-TGCGCTGGATGTATGGGGTCA).

Taqman Analysis of ApoAIV mRNA
Total RNAs were prepared from HepG2 cells by a QIAGEN (Valencia, CA) RNA prep kit, after which the RNA was subjected to reverse transcription reactions using Omniscript RT Kit (QIAGEN) according to the manufacturer’s directions. The resulting cDNA was amplified using TaqMan 2x PCR Master Mix (Applied Biosystems, Foster City, CA). PCR primers and probe for human ApoAIV were designed using Primer Express 1.0 software program (PerkinElmer, Foster City, CA). The sequences for forward primer, reverse primer, and probe were as follows: 5'-CAGCCTGGCTCCCTATGCT-3', 5'-GG-AAGGTCAGGCCCTCAAG-3', 6FAM-CACGCAGGAGAAGC-TCAACCACCA-TAMRA. The PCR products were detected in real time using an ABI-7900HT Sequence Detection System (Applied Biosystems). Each sample was run in triplicate and the relative mRNA level was calculated using a standard curve after normalization with 18S signal representing the mean ± SD of triplicate values after normalization.

Nuclear Run-On Analysis
The nuclei of HepG2 cells were isolated according to the previously described procedure (48). The elongation reaction was carried out as described (49). Human ApoAIV DNA probes were denatured in 0.1 N NaOH for 30 min at room temperature, neutralized in 6x saline sodium citrate (SSC), and applied to Hybond-N membranes (10 µg per slot) using a slot-blot apparatus. 32P-labeled RNA (1–4 x 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% sodium dodecyl sulfate, 1x Denhardt’s (0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% BSA), and 250 µg/ml tRNA at 45 C for 24 h. Membranes were washed four times for 5 min each in 2x SSC at room temperature, incubated in 2x SSC containing 10 µg/ml ribonuclease A for 30 min at 37 C, then washed twice for 30 min each in 0.5x SSC, 0.1% sodium dodecyl sulfate at 65 C. The signal was detected by autoradiography, and quantitated by a phosphorimager (Fuji, Stamford, CT).

Nuclear Protein Isolation
Nuclear protein isolation was done after a modified method described by (50). Briefly, HepG2 cells were treated with 1 µM T0901317 and 1 µM 9-cis-RA for 24 h, and then harvested in Dulbecco’s PBS. After a brief centrifugation, cell pellets were resuspended in buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 1 mM NaF, 1 mM phenymlethylsulfonyl fluoride, and leupeptin]. After that, an aliquot of 10% Nonidet P-40 was added to lyse the cells. After another brief centrifugation, the nuclear pellets were resuspended in ice-cold buffer C [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM NaF, 1 mM phenymlethylsulfonyl fluoride, leupeptin, ß-glycerophosphate, and Na vanadate]. After vigorous vortex and incubation on ice for 30 min, the nuclear extract was then centrifuged. The supernatant, which contained nuclear proteins was subjected to protein quantitation and frozen in aliquots in –80 C.

EMSA
All oligonucleotide LXRE probes were labeled by Klenow fill-in using [{alpha}-32P] deoxy-CTP (NEN, Boston, MA), after the annealing of sense and antisense oligonucleotides. All the oligonucleotides used in EMSA were shown in Table 1Go. Each binding reaction was set up in a final volume of 20 µl, containing 10 mM HEPES (pH 7.5), 75 mM KCl, 1 mM EDTA, 0.05% Triton, 10% glycerol, 1 mM DTT, 2 µg poly[d(I-C)] (Pharmacia) and 4 µg of nuclear protein. After 30 min incubation on ice, 1 µl of [32P]-labeled potential LXRE probe (about 100,000cpm) was added. After another 30 min incubation on ice, samples were subjected to 5% native polyacrylamide gel electrophoresis containing 1x TBE buffer and 5% glycerol. The gel was then vacuum-dried and the results were visualized by autoradiography. For supershift studies, 5 µg each of LXR or RXR antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were included in the incubation for an additional 30 min on ice before electrophoresis. The procedure of gel shift study using in vitro-translated LXR and RXR was described (13).

Construction of Luciferase Reporter Constructs
Human apoAIV gene proximal promoter was cloned by pfu turbo (Stratagene, La Jolla, CA) PCR using 5'-GATCACGCGTCTTCTTTAATGTACTGAACC and 5'-AGTCAGA-TCTTCACCTGCGCTGCAGTGGGA as primers. The PCR fragment was then cloned into pGL-basic (Promega, Madison, WI) vector containing firefly luciferase reporter at BglII and MluI sites to generate HuAIVpromoter-Luc construct. The mutagenesis of the potential LXRE site was carried out on HuAIVpromoter-Luc by QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s protocol. The oligonucleotides used for the mutagenesis were shown as follows: 5'-CCACGTAGTCTCAGGAACACAAAAGAACAAGAGGCCTCTTGGG and 5'-CCCAAGAGGCCTCTTGTTCTTTTGTGTTCCTGAGACTACGTGG. Potential LXREs and their mutant forms were cloned into NheI site of pTAL-Luc (CLONTECH) containing the Herpes simplex virus-TK promoter. The sense strands of these LXREs were summarized in Table 1Go. All constructs were verified by sequencing. All TK-LXRE constructs contain two copies of the potential LXREs. Control LXRE was used as a positive control in experiments and the positive control TK construct has three copies of control LXREs.

Reporter Gene Study
Transfections of various luciferase constructs with or without LXR/RXR constructs intoHepG2 cells were performed in 96-well plates with FuGENE 6 reagent according to the manufacturer’s instructions (Roche Diagnostics Corp., Indianapolis, IN). Cells were 70–80% confluent at the time of the transfection. A mixture of 50 ng of the luciferase construct, 16.7 ng LXR/RXR constuct (under cytomegalovirus promoter in pCMV6 vector) or 33.3 ng pcDNA3 plasmid, as well as 8.3 ng CMV-ß-Gal plasmid was added to each well of the transfected cells. Twenty-four hours after transfection, cells were treated with either 1 µM T0901317 or 1 µM 9-cis-RA or the combination of the two compounds for another 24 h. Cells were then lysed by 1x lysis buffer (PharMingen, San Diego, CA). One fourth of the cell lysate was used to perform ß-Gal assay using CPRG as the substrate (Roche). The rest of the lysate was used to perform luciferase assay using substrate A and B (PharMingen), and Fusion {alpha} plate reader (PerkinElmer). All transfections and subsequent steps were performed in triplicate, and results were normalized by ß-Gal readings.


   ACKNOWLEDGMENTS
 
We would like to thank Dr. Laura Michael and Robert Schmidt for help, Dr. Tom Burris for LXR and RXR expression vectors and Dr. Charlie Bisgaier for apoAIV antibodies. We are also indebted to Drs. Timothy Grese and Steve Kuo-Long Yu for assistance. We would also like to thank Richard Tielking, Jack Cochran, Phyllis Cross, and Patrick Forler for their invaluable technical assistance.


   FOOTNOTES
 
This work was supported by Eli Lilly & Company, and X.-C.J. was supported by National Institutes of Health Grants HL-69817 and HL-64735.

Abbreviations: ABCA1, ATP binding cassette transporter A1; apoAIV, apolipoprotein AIV; ApoE and C, apolipoproteins E and C; DR-4, direct repeat of nuclear hormone-responsive elements separated by four nucleotides; DTT, dithiothreitol; FPLC, fast protein liquid chromatography; HDL, high-density lipoprotein; LXR, liver X receptor; LXRE, LXR-responsive element; RXR, retinoid X receptor; RA, retinoic acid; SSC, saline sodium citrate; TK, thymidine kinase.

Received for publication December 10, 2003. Accepted for publication April 28, 2004.


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 ABSTRACT
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 RESULTS
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 MATERIALS AND METHODS
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Ligands:   22α-Hydroxycholesterol  |  T0901317  |  9-cis-Retinoic acid



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