A Nuclear Receptor Corepressor–Dependent Pathway Mediates ... · of nuclear receptor corepressor complexes was inhibited by LXR ligand treatment, maintaining the CRP gene in a repressed

A Nuclear Receptor Corepressor–Dependent PathwayMediates Suppression of Cytokine-Induced C-Reactive

Protein Gene Expression by Liver X ReceptorFlorian Blaschke, Yasunori Takata, Evren Caglayan, Alan Collins, Peter Tontonoz,

Willa A. Hsueh, Rajendra K. Tangirala

Abstract—C-reactive protein (CRP), the prototypical human acute phase protein, is an independent risk predictor of futurecardiovascular events, both in healthy individuals and in patients with known cardiovascular disease. In addition,previous studies indicate that CRP might have direct proatherogenic properties. Ligand activation of the liver X receptor(LXR), a member of the nuclear hormone receptor superfamily, inhibits inflammatory gene expression in macrophagesand attenuates the development of atherosclerosis in various animal models. We demonstrate herein that 2 syntheticLXR ligands, T0901317 and GW3965, inhibit interleukin-1�/interleukin-6–induced CRP mRNA and protein expressionin human hepatocytes. Knockdown of LXR�/� by short interfering RNAs completely abolished the inhibitory effect ofthe LXR agonist T0901317 on cytokine-induced CRP gene transcription. Transient transfection experiments with5�-deletion CRP promoter constructs identified a region from �125 to �256 relative to the initiation site that mediatedthe inhibitory effect of LXR ligands on CRP gene transcription. Depletion of the nuclear receptor corepressor by specificshort interfering RNA increased cytokine-inducible CRP mRNA expression and promoter activity and reversed LXRligand–mediated repression of CRP gene transcription. Chromatin immunoprecipitation assays indicated that nuclearreceptor corepressor is present on the endogenous CRP promoter under basal conditions. Cytokine-induced clearanceof nuclear receptor corepressor complexes was inhibited by LXR ligand treatment, maintaining the CRP gene in arepressed state. Finally, treatment of C57Bl6/J mice with LXR ligands attenuated lipopolysaccharide-induced mouseCRP and serum amyloid P component gene expression in the liver, whereas no effect was observed in LXR�� knockoutmice. Our observations identify a novel mechanism of inflammatory gene regulation by LXR ligands. Thus, inhibitionof CRP expression by LXR agonists may provide a promising approach to impact initiation and progression ofatherosclerosis. (Circ Res. 2006;99:0-0.)

Key Words: C-reactive protein � liver X receptor � nuclear receptor corepressor

There is compelling evidence that inflammatory processesare key mechanisms in all phases of the atherosclerotic

process, from lesion initiation to progression and, ultimately,the thrombotic complications of atherosclerosis, such asmyocardial infarction and stroke.1,2 Numerous studies haveshown that high-sensitivity C-reactive protein (CRP) levelsindependently predict risk of future cardiovascular events inboth healthy individuals and in patients with establishedcoronary artery disease (CAD).3,4 Moreover, the predictivevalue of high-sensitivity CRP for cardiovascular events inindividuals initially free of reported CAD was significantlyhigher than that associated with other CAD risk markers suchas low-density lipoprotein (LDL) cholesterol or lipoprotein(a).5 Beyond the ability of high-sensitivity CRP to predict therisk of future cardiovascular events, previous studies suggest

that CRP may have direct proatherogenic properties. Forexample, CRP was found to upregulate expression of adhe-sion molecules on endothelial cells6; participate in foam cellformation by mediating LDL uptake by macrophages7 andactivate the complement system both in vivo8 and in vitro.9

Furthermore, our group recently demonstrated that CRPinduces apoptosis in human vascular smooth muscle cells,10

which may lead to increased plaque thrombogenicity andinstability, resulting in an acute cardiovascular event. Al-though beneficial functions of CRP have been reported, suchas upregulation of complement/inhibitory proteins in endo-thelial cells,11 decrease of P-selectin expression in platelets12

and inhibition of neutrophil migration, the published evidencesuggests a proatherogenic effect of CRP. The contradictoryeffects of CRP may be related to bacterial endotoxin or other

Original received July 17, 2006; revision received October 30, 2006; accepted November 8, 2006.From the Division of Endocrinology, Diabetes and Hypertension (F.B., Y.T., E.C., A.C., W.A.H., R.K.T.), David Geffen School of Medicine,

University of California, Los Angeles; Department of Molecular and Genetic Medicine (Y.T.), Ehime University Graduate School of Medicine, Japan;and Howard Hughes Medical Institute, Molecular Biology Institute, and Department of Pathology and Laboratory Medicine (P.T.), University ofCalifornia, Los Angeles.

Correspondence to Rajendra K. Tangirala, PhD, Assistant Professor of Medicine, Division of Endocrinology, Diabetes and Hypertension, David GeffenSchool of Medicine, University of California, Los Angeles, CA 90095. E-mail [email protected]

© 2006 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/01.RES.0000252878.34269.06

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contaminants of CRP or may be explained by the existence of2 distinct conformations of CRP, the native pentamer and amodified, monomeric form.13 Clinical trials are currentlyunderway to determine whether CRP expression may poten-tially provide a novel pharmacological target for treatmentand prevention of atherosclerosis.

CRP is primarily synthesized in hepatocytes, although agrowing body of evidence indicates extrahepatic productionof CRP, such as in macrophages, adipose tissue or endothelialor smooth muscle cells.14–17 In human hepatoma Hep3B cells,CRP gene expression was found to be modestly induced byinterleukin (IL)-6, whereas IL-1�, which alone has no effecton CRP transcription, synergistically increases IL-6–inducedCRP synthesis.18 The transcription factors STAT3 (SignalTransducer and Activator of Transcription 3), nuclear factor�B and members of the C/EBP family (CCAAT box/Enhancer-Binding Protein) participate in cytokine-inducedtranscriptional activation of the CRP gene.19,20 In addition,hepatocyte nuclear factor-1 (HNF-1), HNF-3, and Oct-1 alsoplay important roles in the regulation of CRP expression.21–23

The liver X receptors � (LXR�) and LXR� (also known asNR1H3 and NR1H2, respectively) are members of thenuclear hormone receptor superfamily and have been sug-gested as potential targets for therapeutic intervention inhuman cardiovascular and metabolic disease.24,25 LXR� ishighly expressed in the liver and at lower levels in macro-phages, intestine, adipose tissue, lung, and kidney, whereasLXR� is ubiquitously expressed.26 LXR� and LXR� areligand-activated transcription factors that form heterodimerswith the retinoid X receptor.27 Endogenous activators ofLXRs are oxidized cholesterol derivates (oxysterols).28 Inaddition to endogenous ligands, a number of synthetic LXRagonists, such as T0901317 and GW3965, have been devel-oped. These compounds activate both LXR� and LXR�.29

Previous studies identified LXRs as important regulators ofreverse cholesterol transport and lipid and glucose metabo-lism.30 In addition LXRs also play a role in innate immunityand regulate inflammatory gene expression in macro-phages.31–33 Indeed, synthetic LXR agonists have been shownto both delay the development of atherosclerosis in geneti-cally prone mouse models and induce regression of preexist-ing atherosclerotic lesions.34–36 In the absence of ligand,heterodimers of LXR and retinoid X receptor, in complexwith corepressors such as nuclear receptor corepressor(NCoR) and the related factor SMRT (Silencing Mediator ofRetinoic acid and Thyroid hormone receptors), are bound toLXR-responsive elements and inhibit target gene transcrip-tion.37 Binding of ligand to LXR causes a conformationalchange that results in release of corepressors and recruitmentof coactivators, leading to induction of target genes. How-ever, the mechanism underlying the repression of inflamma-tory genes by LXR is poorly understood. Previous studiessuggest that indirect mechanisms, such as competition fortranscriptional coactivators and antagonism of the nuclearfactor �B signaling pathway, are involved.38

The role of LXR in regulating the expression of acutephase proteins in the liver has not yet been investigated. In thepresent study, we demonstrate that LXR ligands inhibitcytokine-induced CRP expression in human hepatocytes.

This effect is, at least partially, mediated by inhibition ofcytokine-induced NCoR clearance from the promoter. Theseobservations define a novel function of LXRs in the controlof liver gene expression and support the potential utility ofLXR ligands to prevent and treat cardiovascular disease.

Materials and MethodsMaterialsPrimary human hepatocytes (PHHs) and hepatocyte culture medium(HCM BulletKit) were obtained from Cambrex Bioscience. Thehuman hepatoma cell line Hep3B and minimum essential medium(MEM) were purchased from American Type Culture Collection.Hybond enhanced chemiluminescence (ECL) nitrocellulose mem-brane and ECL Western blotting detection reagents were purchasedfrom Amersham Life Science, and horseradish peroxidase–linked anti-rabbit and anti-mouse antibodies were obtained from Cell Signaling.Recombinant human IL-1� and human IL-6 were from R&D Systems.Lipopolysaccharide (LPS) and T0901317 were purchased from Sigma-Aldrich, GW3965 was kindly provided by Dr Timothy Willson (Glaxo-SmithKline Inc). Antibodies were commercially obtained from thefollowing suppliers: CRP (clone CRP-8) and anti–FLAG M2 fromSigma-Aldrich; NCoR from Affinity BioReagents.

Cell CultureHuman hepatoma Hep3B cells were cultured in MEM supplementedwith 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.PHHs were maintained in HCM supplemented with ascorbic acid,BSA-FAF, transferrin, insulin, hEGF and GA-1000 according to theinstructions of the manufacturer. For ligand treatment, Hep3B cellswere serum-deprived in 0% FBS MEM and treated with T0901317or GW3965 for 18 hours before stimulation with IL-1� and IL-6, ata final concentration of 20 ng/mL and 10 ng/mL, respectively. PHHswere treated with LXR ligands in HCM supplemented with growthfactors for 18 hours before stimulation with IL-6 (10 ng/mL). For alldata shown, individual experiments were repeated at least 3 timeswith different lots of cells.

RNA Isolation and Northern BlottingTotal RNA isolation and Northern blotting were performed aspreviously described.39 Human CRP cDNA was obtained from DrAlok Agrawal (East Tennessee State University, Johnson City).Blots were cohybridized with human GAPDH cDNA (Sigma-Al-drich) to assess equal loading of samples.

Western Blot AnalysisCells were harvested at the indicated time points and sonicated insolubilization buffer (Cell Signaling). Supernatants of Hep3B cellswere collected as indicated and concentrated using centrifugal filterunits (Millipore). Nuclear extracts were isolated using the NuclearExtraction Kit (Panomics) according to the instructions of themanufacturer. Western blot analyses were performed as previouslydescribed.39

Reverse Transcription and Quantitative Real-TimePolymerase Chain ReactionTotal RNA was isolated using the RNeasy MINI Kit (QIAGEN).Total RNA (400 ng) was reverse transcribed with random hexamersusing the TaqMan Reverse Transcription Reagent Kit (AppliedBiosystems) according to the instructions of the manufacturer.Real-time quantitative polymerase chain reaction (PCR) assays wereperformed by using an ABI-PRISM 7700 system (Applied Biosys-tems) in a total volume of 25 �L. Each sample was analyzed intriplicate, and cycle thresholds of individual genes were normalizedto corresponding GAPDH mRNA expression values. Primer andprobes used in the experiments were obtained from AppliedBiosystems.

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Immunofluorescence MicroscopyHep3B cells, grown on 2-well chamber-slides (Becton Dickinson),were serum-deprived in the presence of LXR ligand or vehicle(DMSO) for 18 hours followed by treatment with IL-1�/IL-6 for anadditional 24 hours. After 12 hours of cytokine stimulation, BDGolgiStop (containing monesin; BD Biosciences) was added to blockthe intracellular protein transport processes. Cells were fixed for 15minutes with 3.7% paraformaldehyde (in PBS) and permeabilized in0.2% Triton X-100 (in PBS) for 5 minutes. After blocking ofnonspecific staining with the Image-iT FX signal enhancer (Molec-ular Probes), cells were stained overnight with a CRP antibody,followed by a secondary goat anti-mouse IgG (1:200; MolecularProbes). Cell nuclei were counterstained with 4�,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI). Cells were visualizedwith a fluorescence microscope, and colocalization of fluoresceinand DAPI staining was performed by overlay projections.

Transient Transfection and Luciferase AssayTransient transfections were performed in triplicate in 6-well plates,using the FuGENE 6 reagent (Roche) according to the instructions ofthe manufacturer. Twenty-four hours after transfection, cells wereincubated in serum-free medium in the presence of vehicle (DMSO)or LXR ligands for 18 hours, followed by cytokine stimulation.Luciferase activity was assayed 24 hours after IL-1� (20 ng/mL) andIL-6 (10 ng/mL) treatment using a Dual Luciferase Reporter AssaySystem (Promega) according to the instructions of the manufacturer.All experiments were repeated at least 3 times with different cellpreparations. The CRP promoter constructs were generously pro-vided by Dr David Samols (Case Western Reserve University,Cleveland, Ohio); the full-length NCoR expression vector (PKCR2-NCoR) was obtained from Dr Anthony Hollenberg (Beth IsraelDeaconess Medical Center and Harvard Medical School, Boston,Mass).

Short Interfering RNAShort interfering RNA (siRNA) specific for NCoR, LXR�, andLXR� (SMARTpool short interfering RNA [siRNA]) and nonsilenc-ing control siRNA were purchased from Dharmacon. Transfectionswere performed with siRNAs (40 nmol/L) using the transfectionreagent DharmaFECT Reagent 4. Forty-eight hours after transfec-tion, cells were serum deprived in the presence of T0901317 orvehicle (DMSO), followed by IL-1�/IL-6 stimulation as indicated.

Chromatin Immunoprecipitation AssayChromatin immunoprecipitation (ChIP) assays were performed us-ing a ChIP assay kit from Upstate according to the instructions of themanufacturer. Briefly, cells were pretreated with 5 �mol/LT0901317 for 18 hours and stimulated with a combination of IL-1�(20 ng/mL) and IL-6 (10 ng/mL) as indicated. Cells were crosslinkedwith 2 mmol/L disuccinimidyl glutarate for 45 minutes beforecrosslinking for 15 minutes with 1% formaldehyde. This 2-stepcrosslinking method has been shown to be more efficient than theconventional single formaldehyde crosslinking procedure.40 Afterlysis, cells were sonicated using a Branson Digital Sonifier model450 (Branson Ultrasonics Corporation). Chromatin fragments wereimmunoprecipitated with an antibody directed against NCoR (2 �g)overnight at 4°C. Rabbit IgG (Santa Cruz) was used as a negativecontrol. DNA fragments were purified from chromatin using theQUIAquick PCR Purification Kit (QIAGEN) according to theinstructions of the manufacturer. The final DNA extractions wereamplified by PCR, using the following primer pairs: forward,5�-CAAAGTGGAGCCCTGAGAGA-3�; reverse, 5�-CTACCTC-CTCCTGCCTGGAT-3�. As control, primers against an unrelatedregion of the CRP promoter were used: forward, 5�-CACCAGCAT-GGCACATGTAT-3�; reverse, 5�-AGCTGCCTCTCCAACACCTA-3�; and forward, 5�-TGGTCTTGACCAGCCTCTCT-3�; reverse,5�-ACAGACTGACCCCTTCTCCA-3�. The PCR products wereresolved on 2% agarose gels and visualized using ethidium bromide.

Analysis of CRP and SAP by ELISAConcentration of CRP in the supernatant of Hep3B cells wasmeasured with a human hsCRP ELISA kit (ALPCO Diagnostics)according to the instructions of the manufacturer. Serum levels ofmurine SAP were determined using a murine SAP ELISA kit(GENTAUR). Samples were assayed in duplicate.

Animal StudiesMale C57Bl6/J mice (The Jackson Laboratory) and LXR��-knockout mice on a mixed background (C57Bl6/J and 129Sv),received daily intraperitoneal injections of T0901317 (20 mg/kgbody weight per day in DMSO) or vehicle (DMSO). At day 4 oftreatment, bacterial LPS (3 mg/kg body weight) was injectedintraperitoneally. Twenty-four hours later, animals were euthanizedand liver samples were harvested. Serum samples were collectedfrom the mice 24 hours after injection of LPS. All animal protocolswere approved by the University of California, Los Angeles AnimalResearch Committee and complied with all federal, state, andindustrial regulations.

Statistical AnalysisANOVA and paired or unpaired t test were performed for statisticalanalysis as appropriate. Probability values less than 0.05 wereconsidered to be statistically significant. Results are expressed asmean�SEM.

ResultsLXR Agonists Suppress CRP Expression inHep3B CellsBased on previous studies, we used the human hepatoma cellline Hep3B as a cell culture model to study CRP generegulation in vitro.41,42 Given their antiatherogenic and anti-inflammatory properties, LXR agonists have been proposedas promising therapeutic approach to treat cardiovasculardisease.38 To determine the effect of synthetic LXR ligandson cytokine-induced CRP expression, quiescent Hep3B cellswere pretreated with T0901317 or GW3965 for 18 hours andstimulated with a combination of IL-1� (20 ng/mL) and IL-6(10 ng/mL) for an additional 24 hours. As shown in Figure1A, both synthetic LXR ligands inhibited IL-1�/IL-6–stim-ulated CRP mRNA expression in a dose-dependent manner,as determined by Northern blotting and quantitative real-timeRT-PCR (76.1% inhibition at 5 �mol/L T0901317 and 69.1%inhibition at 5 �mol/L GW3965 versus IL-1�/IL-6 alone;P�0.05). LXR agonist–mediated inhibition of CRP mRNAexpression also correlated with reduced levels of CRP proteinexpression, as analyzed by immunofluorescence staining,Western blot analyses, and ELISA. As shown in Figure 1B,no cellular CRP protein expression could be detected inquiescent Hep3B cells by immunofluorescence, whereasstimulation with IL-1� (20 ng/mL)/IL-6 (10 ng/mL) resultedin profound cytoplasmic staining. Pretreatment with twodifferent LXR ligands, T0901317 and GW3965 (both at5 �mol/L), inhibited cytokine-induced CRP protein expres-sion. Similarly, cytokine-induced CRP protein release to theculture supernatant was suppressed by LXR ligands in adose-dependent manner (Figure 1C). Together, these datademonstrate that LXR ligands inhibit cytokine-induced CRPmRNA and protein expression in Hep3B cells.

LXR Ligands Inhibit CRP Expression in PHHsWe next investigated whether the inhibitory effect of LXRagonists on cytokine-induced CRP expression is also ob-

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Figure 1. The synthetic LXR agonists T0901317 and GW3965 inhibit cytokine-induced CRP mRNA and protein expression in Hep3Bcells. Hep3B cells were pretreated in MEM medium containing 0% FBS for 18 hours with vehicle (DMSO) or the indicated concentra-tions of LXR ligands. Cells were then stimulated with a combination of IL-1� (20 ng/mL) and IL-6 (10 ng/mL) for an additional 24 hours.A, Total RNA was isolated and CRP mRNA expression was measured by Northern blotting and quantitative real-time PCR and normal-ized to GAPDH mRNA expression. Data are presented as mRNA levels relative to untreated control (Con). B, CRP protein expressionwas determined by immunofluorescence staining. CRP was labeled with a monoclonal anti-CRP antibody, followed by a fluorescein-conjugated (green) secondary antibody. The cell nuclei were stained with DAPI (blue). C, CRP content in cell supernatants was assayedby Western blotting using a CRP-specific antibody and ELISA. Quantification was performed from 3 independently performed experi-ments. Results are presented as mean�SEM. *P�0.05 vs IL-1�/IL-6–stimulated cells.




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served in PHHs. Cells were stimulated with 10 ng/mL IL-6alone, because, in contrast to Hep3B cells, IL-6–stimulatedCRP expression is reduced by costimulation with IL-1�.43 Asdepicted in Figure 2, preincubation of PHHs with eitherT0901317 or GW3965 resulted in a significant suppression ofIL-6–induced (10 ng/mL) CRP mRNA expression.

LXR Activation Suppresses Cytokine-InducedCRP TranscriptionThe mechanism for the inhibition of cytokine-induced CRPtranscription by LXR ligands is unlikely to be direct, asactivation of LXR by its ligands has not been reported tofunction as a transcriptional repressor. Moreover, sequenceanalysis of the 5�-flanking region of the CRP promoter didnot reveal the presence of potential LXR response elements.To identify the promoter elements that mediate the transcrip-tional suppression of CRP in Hep3B cells by LXR ligands,we used a series of 5�-deletion constructs (Figure 3A). Wefound, as previously reported, that the effect of IL-1�/IL-6stimulation was significantly lower in deletion �125CRP-Luc as compared with longer promoter constructs and thatinducibility by cytokines was lost in the �55CRP-Luc and�86CRP-Luc constructs.41 Moreover, although both the�256CRP-Luc and �125CRP-Luc constructs retained cyto-kine inducibility, only the �256CRP-Luc construct demon-strated transcriptional repression by the LXR ligandT0901317. These findings indicate that the ability of LXR toinhibit the CRP promoter is dependent on transcription factorbinding sites located between �256 and �125 relative to thetranscription initiation site. We also performed promoterassays using various concentrations of LXR ligand. Asdemonstrated in Figure 3B, T0901317 dose-dependentlyattentuated IL-1�/IL-6–induced CRP promoter activity overa concentration range of 1 to 5 �mol/L.

LXR Ligand-Mediated Inhibition of CRPExpression Is Receptor DependentTo address whether the inhibitory effect observed withT0901317 is mediated by LXRs, we used siRNAs to knock-

down LXR� and LXR�. Inhibition of LXR�/� expression inHep3B cells using specific siRNAs completely abolished theability of T0901317 (2 �mol/L) to inhibit cytokine-inducedCRP mRNA expression (Figure 4A) and promoter activity(Figure 4B). In addition, cytokine-induced CRP gene tran-scription and promoter activity was enhanced in Hep3B cellstransfected with the specific siRNA compared with controlsiRNA transfected cells. Taken together, these data demon-strate that LXR ligands repress CRP through a receptordependent mechanism.

Inhibition of CRP Transcription Activity by NCoRRecent studies indicate that NCoR complexes are involved inbasal suppression of inflammatory response genes in macro-phages, with loss of NCoR resulting in derepression ofnuclear factor �B and activator protein-1 target genes.44,45

This interaction between NCoR and inflammatory genesprompted us to address the role of NCoR in the regulation ofcytokine-induced CRP transcription in Hep3B cells. There-fore, we transiently transfected the �904CRP-Luc promoterconstruct along with an expression vector for NCoR. Nuclearlocalization of overexpressed NCoR was confirmed by indi-rect immunofluorescence (Figure 5A). In accordance withprevious observations,46 NCoR-transfected cells displayed acharacteristic nuclear speckle staining (Figure 5A), whereasan IgG control antibody exhibited only background staining(data not shown). As shown in Figure 5B, cotransfection ofthe NCoR expression vector resulted in a dose-dependentinhibition of IL-1�/IL-6–induced CRP promoter activity,indicating that NCoR participates in regulation of CRPtranscriptional activity.

NCoR-Specific siRNA Reverses LXR-MediatedInhibition of CRP TranscriptionWe next used a NCoR-specific siRNA to evaluate whetherinhibition of NCoR expression results in reversal of repres-sion of CRP gene transcription by LXR ligands. Efficacy ofNCoR knockdown by siRNA was evaluated by Western blotanalyses of NCoR protein expression in Hep3B cells trans-

Figure 2. LXR activation inhibits cytokine-induced CRP gene transcription in PHHs. Cells were pretreated for 18 hours with vehicle(DMSO) or LXR ligands as indicated. Subsequently, cells were either nontreated or treated with IL-6 (10 ng/mL) for 24 hours. Cells werethen harvested and analyzed for CRP mRNA expression by quantitative real-time PCR. Results were normalized to corresponding GAPDHmRNA expression values. Data are expressed as fold induction�SEM over unstimulated control (Con). *P�0.05 vs IL-6–treated cells.




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Figure 3. LXR activation inhibits CRP promoter activity. Hep3B cells were transiently transfected with CRP promoter constructs as described inMaterial and Methods. A, After transfection, cells were incubated with either vehicle (DMSO) or T0901317 (5 �mol/L) for 18 hours and stimulatedwith IL-1� (20 ng/mL)/IL-6 (10 ng/mL) for an additional 24 hours. B, Transfected Hep3B cells were pretreated with vehicle (DMSO) or the indicatedconcentrations of T0901317, followed by stimulation with a combination of IL-1� (20 ng/mL) and IL-6 (10 ng/mL). Luciferase activity was assayed 24hours after cytokine treatment. All experiments were repeated at least 3 times. Data are expressed as normalized luciferase activity relative tounstimulated control and presented as mean �SEM. *P�0.05 vs vehicle, #P�0.05 vs IL-1�/IL-6–stimulated cells.




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fected with the specific siRNA and a control siRNA (Figure6A). Inhibition of NCoR expression using a NCoR-specificsiRNA resulted in attenuation of LXR ligand–mediated inhi-bition of CRP mRNA transcription compared with controlsiRNA-transfected cells (53.1% inhibition versus 80.2% in-

hibition at 5 �mol/L T0901317; P�0.05; Figure 6B). Con-sistent with these findings, knockdown of NCoR expressionreversed LXR ligand–mediated CRP promoter repression(47.6% inhibition versus 77.5% inhibition at 5 �mol/LT0901317; P�0.05; Figure 6C). Taken together, these data

Figure 4. LXR ligands mediate CRPgene repression through a receptor-dependent mechanism. A, Hep3B cellswere transfected either with scrambledsiRNA or LXR�/� siRNA. Forty-eighthours after transfection, cells were serumdeprived in the presence of vehicle(DMSO) or the LXR agonist T0901317.Cells were stimulated with IL-1� (20ng/mL)/IL-6 (10 ng/mL) for 24 hours, andtotal RNA was analyzed for CRP mRNAexpression by quantitative real-timePCR. B, Hep3B cells were transientlytransfected with the Luc-904 CRP pro-moter construct and the indicated siR-NAs. Following transfection, cells weretreated as described in A and luciferaseactivities were analyzed. Experimentswere performed in triplicate and data arepresented as mean�SEM. *P�0.05 vsvehicle, #P�0.05 vs IL-1�/IL-6–stimu-lated cells.

Figure 5. Overexpression of NCoR inhib-its cytokine-induced CRP promoteractivity. Hep3B cells were cotransfectedwith a FLAG-tagged full-length NCoRexpression vector and the Luc-904 CRPpromoter construct as indicated. A,Using a monoclonal anti–FLAG M2 anti-body, followed by a fluorescein-conjugated (green) secondary antibody,NCoR protein was detected in thenucleus, but not cytoplasm of trans-fected cells. The cell nuclei were stainedwith DAPI (blue). B, After transfection,cells were incubated in 0% FBS MEMmedium for 18 hours, followed by stimu-lation with a combination of IL-1� (20ng/mL) and IL-6 (10 ng/mL) for 24 hours.Luciferase activities were assayed asdescribed in Figure 4. Results areexpressed as fold induction vs untreatedcontrol and presented as mean�SEM.*P�0.05 vs vehicle.




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indicate that inhibition of CRP gene transcription is mediated,at least in part, through a NCoR dependent mechanism.

LXR Activation Prevents Cytokine-InducedDissociation of NCoR From the CRP PromoterOur previous observations predicted that NCoR complexesassociate with the CRP promoter. To confirm that NCoR

binds to the endogenous CRP promoter and that LXR ligandsinterfere with this binding in vivo, we next performed ChIPexperiments. PCR amplification using primer pairs that covera region from �6 to �241 in the CRP promoter demonstratedthat NCoR is present on this CRP promoter sequence underbasal conditions (Figure 7). Time-course experiments re-vealed that NCoR was cleared from the CRP promoter within60 minutes of IL-1�/IL-6 treatment. However, clearance ofNCoR was substantially inhibited by T090137 treatment.ChIP analysis with primers against an unrelated CRP pro-moter region revealed no NCoR binding. Together, these datademonstrate that LXR ligands prevent cytokine-induced dis-sociation of NCoR from the CRP promoter, thus maintainingthe CRP gene in a repressed state.

LXR Agonists Inhibit Acute Phase Response GeneExpression in VivoAlthough CRP is the major acute phase protein in humansand most mammals, serum amyloid P component (SAP) is themajor acute-phase protein in the mouse.47 To finally deter-

Figure 6. siRNA targeting NCoR reverses LXR ligand–mediatedrepression of CRP gene transcription. A, Efficacy of NCoRsiRNA was evaluated by transfecting Hep3B cells with the indi-cated NCoR-specific and control siRNA as described in Materialand Methods. Nuclear protein levels were determined by West-ern blot analyses using a NCoR-specific antibody. B, Hep3Bcells were transfected with control siRNA or a NCoR-specificsiRNA. Following transfection, cells were pretreated for 18 hourswith vehicle (DMSO) or LXR ligand before stimulation with IL-1�(20 ng/mL)/IL-6 (10 ng/mL) for 24 hours. Total RNA was iso-lated, and the level of CRP mRNA was examined by quantitativereal-time PCR. C, Cells were transiently transfected with theLuc-904 CRP promoter construct and the indicated siRNAs asdescribed in Material and Methods. Forty-eight hours aftertransfection, cells were treated as described in B and luciferaseactivity was measured. Experiments were performed in tripli-cate; data are expressed as fold induction vs control and pre-sented as mean�SEM. *P�0.05 vs vehicle, #P�0.05 vs IL-1�/IL-6–stimulated cells.

Figure 7. LXR ligand prevents cytokine-induced dissociation ofNCoR from the CRP promoter. ChIP assays were performedusing chromatin isolated from serum-deprived Hep3B cells pre-treated for 18 hours with vehicle (DMSO) or T0901317 (5 �mol/L), followed by stimulation with IL-1� (20 ng/mL)/IL-6 (10ng/mL). Crosslinked cell lysates were subjected to immunopre-cipitation with rabbit IgG as control (lane 1) or a NCoR-specificantibody (lanes 2 to 6). DNA precipitates were isolated and thensubjected to PCR using primers corresponding to the regionfrom �241 to �6 of the CRP gene (a). Input DNA was detectedby using 10% of the soluble chromatin before immunoprecipita-tion. PCR with primers against an unrelated promoter regionupstream (b) and downstream (c) from �241 to �6 revealed noNCoR binding. The data shown is representative of 3 indepen-dently performed experiments.




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mine whether the effects of LXR ligands to suppress acutephase response gene expression are applicable in vivo, thesynthetic LXR agonist T0901317 (20 mg/kg body weight perday) or vehicle (DMSO) was administered to C57Bl6/J andLXR�� knockout mice 3 days before intraperitoneal LPSinjection (3 mg/kg body weight). Twenty-four hours afterLPS treatment animals were euthanized. Hepatic mRNAexpression levels of SAP and CRP in LXR ligand–treatedC57Bl6/J mice were significantly decreased compared withthose in vehicle-treated mice (60.2% and 76.9% inhibitionversus LPS alone; n�4, P�0.05; Figure 8). At 24 hours afterinjection of LPS, serum levels of SAP, as determined byELISA, were significantly reduced in C57Bl6/J mice treatedwith T0901317 (38.6% inhibition versus LPS alone; n�4,P�0.05; Figure 8B). The ability of the LXR agonistT0901317 to inhibit LPS-induced acute phase response geneexpression is strictly receptor-dependent, because no effecton CRP and SAP mRNA (Figure 8A) and SAP serum levels(Figure 8B) was observed in ligand-treated LXR��/� mice.Taken together, these results show that LXR agonists inhibitacute-phase response gene expression in vivo and furtheremphasize the important role of LXRs as modulators ofinflammatory gene response both in vitro and in vivo.

DiscussionLXR ligands play a dual role in maintaining lipid andlipoprotein homeostasis and in modulating inflammatorygene expression in both macrophages and lymphocytes.32,48,49

In animal models of atherosclerosis, such as apolipoproteinE–deficient (apoE�/�) and LDL receptor–deficient(LDLR�/�) mice, we found that LXR ligand treatment re-duced the development of atherosclerosis.34 Moreover, recentstudies revealed that the LXR signaling pathway links innateimmunity to macrophage cholesterol metabolism.50 Thus, theability of LXRs to promote reverse cholesterol transport,attenuate inflammation, and improve glucose tolerance iden-tifies the LXR pathway as a potential target for noveltherapeutic interventions in human cardiovascular dis-ease.38,51,52 In the present study, we demonstrate that LXRligands attenuate cytokine-induced CRP expression in bothHep3B cells and PHHs. This effect is, at least partially,mediated by inhibition of NCoR clearance from the CRPpromoter, which prevents the switch from active repression totranscriptional activation. The observations described hereinidentify a novel mechanism by which LXR agonists inhibitinflammatory gene responses and expand the role for LXRagonists as antiinflammatory ligands to hepatocytes.

The human hepatoma cell line Hep3B is a widely usedmodel for studying the regulation of CRP gene transcription.The major inducer of CRP gene transcription in Hep3B cellsis IL-6; however, maximal gene expression is achieved onlywhen both IL-1� and IL-6 are present.41 The results presentedhere demonstrate that LXR ligands are negative regulators ofCRP gene expression in hepatocytes. In both Hep3B cells andprimary hepatocytes, cytokine-induced CRP mRNA and pro-tein expression was significantly inhibited by the 2 syntheticLXR agonists, T0901317 and GW3965. siRNA-mediateddepletion of LXR�/� abolished the inhibitory effect ofT0901317 on CRP gene expression, demonstrating a

receptor-dependent mechanism. However, analysis of theCRP promoter did not reveal the presence of any putativeLXR-responsive elements, indicating that the inhibition ofcytokine-induced CRP transcription by LXR ligands is indi-rect through inhibition of binding of other transcriptionfactors, competition for transcriptional activators, or recruit-ment of corepressor complexes. Previous studies have high-lighted important roles for various transcription factors, suchas STAT3, C/EBP-�/�, HNF-1�, HNF-3, or Oct-1 in regu-lating CRP gene expression.19,21–23 To localize the regions

Figure 8. The LXR agonist T0901317 inhibits LPS-induced APRgene expression in the liver in vivo. C57Bl6/J and LXR�� knock-out (DKO) mice were pretreated with T0901317 (20 mg/kg perday) or vehicle (DMSO) for 4 days, followed by intraperitonealinjection of saline or LPS (3 mg/kg body weight), respectively. A,Mouse CRP and SAP mRNA expression in the liver were ana-lyzed by quantitative real-time PCR. Relative mRNA expressionlevels were determined after normalization to internal controlGAPDH mRNA levels. B, Serum levels of SAP in the mice weredetermined at 24 hours after injection of saline or LPS. Resultsare presented as mean�SEM. *P�0.05 vs vehicle treated mice,#P�0.05 vs LPS injected mice (n�4 mice per group).




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important for LXR-dependent repression, we used a series of5�-deletion constructs. We identified a region in the CRPpromoter between �256 and �125 that is essential for LXRligand–mediated inhibition of cytokine-induced transcrip-tional activity. This promoter region contains a binding sitefor HNF-1�. Previous studies have shown that mutations ineither of these 2 HNF-1 sites abolished CRP gene induction.21

In addition, HNF-1� overexpression induced CRP promoteractivity in Hep3B cells in the absence of IL-1�/IL-6, whereastransfection with a dominant negative HNF-1� expressionvector had no effect (data not shown). However, despite theimportant role of the HNF-1� site for the transcriptionalregulation of the CRP gene, electrophoretic mobility-shiftassays revealed no significant changes in protein/DNA com-plexes in the presence of LXR ligands (data not shown).

This observation prompted us to investigate the role ofcorepressors in LXR ligand–mediated inhibition of CRP genetranscription. Previous studies demonstrate that NCoR exertsrepressive effects on basal inflammatory gene expression byinteracting with several classes of DNA-binding transcriptionfactors,53–55 with loss of NCoR resulting in gene transcriptionin macrophages.45 Recently, Pascual et al defined a novelpathway by which peroxisome proliferator-activated receptor� (PPAR-�) represses inflammatory gene responses in mac-rophages.44 PPAR-� ligand–induced SUMOylation targetsPPAR-� to NCoR-containing complexes associated withinflammatory gene promoters in the basal state. This in turnprevents LPS-mediated clearance of NCoR complexes fromthe promoter by inhibiting the recruitment of the ubiquityla-tion/19S proteosome machinery. We thus explored the pos-sibility that the inhibitory effect on CRP expression observedwith LXR ligands is mediated by NCoR. Overexpression ofNCoR resulted in a potent inhibition of cytokine-inducedCRP promoter activity, suggesting that NCoR plays animportant role in regulating CRP gene transcription. Consis-tent with these findings, knockdown of NCoR using aNCoR-specific siRNA further increased cytokine-inducedCRP gene transcription. Furthermore, inhibition of NCoRexpression by siRNA reversed LXR ligand–dependent inhi-bition of CRP mRNA expression and promoter activity.These findings suggest that NCoR complexes associate withthe CRP promoter in the basal state. ChIP assays revealed thatNCoR was bound to the CRP promoter under basal condi-tions. Moreover, cytokine stimulation cleared NCoR from thepromoter, which was inhibited by treatment of cells with theLXR ligand. These studies suggest that the presence of NCoRcomplexes on the promoter maintains the CRP gene in arepressed state, preventing the switch from active repressionto transcriptional activation. Additional studies are necessaryto elucidate in more detail the LXR ligand–mediated inhibi-tion of NCoR clearance from the CRP promoter and toidentify the transcription factors interacting with the NCoRcorepressor complex.

We further addressed whether LXR� or LXR� expressionin hepatocytes is regulated in response to LXR ligands.LXR� is highly expressed in the liver, whereas LXR� isubiquitously expressed.26 Whereas treatment of Hep3B cellswith LXR ligands had no effect on LXR� mRNA expression,LXR agonists dose-dependently induced LXR� mRNA (Fig-

ure I in the online data supplement). Similar to the effect onLXR� expression, the LXR target gene ABCA1 was upregu-lated by LXR agonists (data not shown). The observedautoregulation of the LXR� gene in liver might have impli-cations for the inhibitory effect of LXR ligands on CRP geneexpression. However, previous studies performed in macro-phages obtained from LXR��/� and LXR��/� mice indicatethat both LXR isoforms are negative regulators of inflamma-tory gene expression.48 The first direct target gene of LXRs tobe identified in mice was Cyp7a1, the rate-limiting enzyme inhepatic bile acid synthesis.56 Subsequent studies demon-strated that activation of hepatic LXR also exerts potentiallybeneficial effects by regulating genes involved in cholesterolmetabolism. Induction of ABCG5 and ABCG8, members ofthe ATP-binding cassette (ABC) superfamily, results inincreased cholesterol excretion into the bile.57 More recently,LXR agonists were found to induce apolipoprotein AIV(apoAIV) transcription in human hepatoma HepG2 cells,suggesting that this effect may contribute to the antiathero-genic effects of LXR activation.58 However, in addition tomodulating cholesterol metabolism, LXRs also induce lipo-genic genes. Mice treated with synthetic LXR agonistsupregulate fatty acid synthase, acetyl-coenzyme A carboxy-lase and the transcription factor SREBP-1c, resulting in anincrease of hepatic and plasma triglycerides.38 The develop-ment of gene- or isoform-specific LXR agonists is a promis-ing approach to induce the desired beneficial effects of LXRactivation without the detrimental hepatic effects.

Numerous studies indicate that CRP, in addition to being arisk marker for future cardiovascular events, plays a directrole in the development and progression of atheroscleroticlesions.59 Thus, approaches to directly target the synthesis ofCRP or blocking the function of CRP by inhibitors might bea promising new tool for both primary prevention andtreatment of cardiovascular disease. Although extrahepaticsynthesis of CRP, such as by macrophages, neuronal cells,adipose tissue, and renal cortical tubular epithelial cells hasbeen shown,15,60–62 the principal source of circulating CRP isthe hepatocyte, which synthesizes CRP under the transcrip-tional control of inflammatory cytokines, in particular IL-6.63

In mice, SAP and serum amyloid A are the major acute-phaseresponse (APR) genes, whereas CRP, because of its modestregulation, is considered a minor APR gene. However,several common molecular properties of SAP and CRP havebeen established. Both are members of the pentraxin family,with a similar pentagonal arrangement of their subunits anddisplay calcium-dependent binding to specific substrates.64,65

In addition, both mouse SAP and human CRP respond toIL-1� and IL-6 stimulation, which enhances binding ofSTAT3 and C/EBP� to their promoters. In contrast, previousfindings indicate that only STAT3 is involved in mouse CRPgene expression rather than a complex, synergistic interactionbetween STAT3 with C/EBP-�.66 These differences in themouse CRP and SAP promoter regions might account for thedifferent transcriptional response during inflammation. Wefound a significant increase in liver CRP and SAP mRNAlevels 24 hours after LPS injection of C57Bl6/J andLXR��/� mice. In C57Bl6/J mice, induction of these APRgenes was significantly inhibited by pretreatment with the




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LXR ligand T0901317. In contrast, the ability of T0901317 toinhibit LPS-induced expression of CRP and SAP was com-pletely abolished in LXR��/� mice, indicating a receptor-dependent mechanism. Thus, our results extend the inhibitoryeffect of LXR ligands on human CRP gene transcription invitro to related APR genes in vivo.

In summary, results presented in this study demonstratethat LXR agonists inhibit cytokine-induced CRP expressionin hepatocytes and negatively interfere with cytokine-inducedNCoR dissociation from the CRP promoter, thus maintainingthe CRP gene in a repressed state. LXR ligand–mediatedattenuation of CRP expression in vitro translates into in vivoas LPS-induced APR gene expression in murine liver isinhibited in LXR agonist–treated animals. Thus, inhibition ofCRP expression by LXR agonists may offer a novel thera-peutic approach for the primary prevention and treatment ofatherosclerotic disease.

AcknowledgmentsWe thank Dr Alok Agrawal and Dr David Samols for providing theCRP cDNA and promoter constructs and Dr Anthony Hollenberg forproviding the NCoR expression vector.

Sources of FundingThis study was supported by NIH grant HL075171 to (W.A.H.). F.B.was supported in part by a research fellowship from Philip MorrisUSA Inc. Y.T. was supported by Japan Heart Foundation/BayerYakuhin Research Grant Abroad. P.T. is an investigator of theHoward Hughes Medical Institute.

DisclosuresNone.

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Hsueh and Rajendra K. TangiralaFlorian Blaschke, Yasunori Takata, Evren Caglayan, Alan Collins, Peter Tontonoz, Willa A.

Cytokine-Induced C-Reactive Protein Gene Expression by Liver X ReceptorA Nuclear Receptor Corepressor-Dependent Pathway Mediates Suppression of

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Online Supplement Blaschke et al. NCoR mediates anti-inflammatory properties of LXR

Supplemental Figure 1. Synthetic ligands for LXR induce LXRα mRNA expression in

Hep3B cells. Cells were incubated for 18 hours in MEM medium containing 0% FBS and

then cultured for additional 48 hours in the presence of vehicle (DMSO) or the LXR

ligands T0901317 or GW3965 as indicated. A, Total RNA (20 µg per lane) was analyzed

by Northern blotting and hybridized with 32P-labeled probes for LXRα. B, LXRα and

LXRβ transcript levels were analyzed by quantitative real-time PCR and normalized to

GAPDH mRNA expression. Data are presented as mRNA levels relative to untreated

control (Con). Experiments were performed in triplicate (*P<0.05 vs unstimulated cells;

mean±SEM).

Supplemental Figure 1, Blaschke et al.

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4 LXRαLXRβ

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4LXRαLXRβ

LXR

α/ β

mR

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Con DMSO 0.1 0.5 1 2 5Con DMSO 0.1 0.5 1 2 5

T0901317 (µM) GW3965 (µM)

Con DMSO 0.1 0.5 1 2 5

T0901317 (µM)

Con DMSO 0.1 0.5 1 2 5

GW3965 (µM)

LXR-alpha LXR-alpha

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A Nuclear Receptor Corepressor–Dependent Pathway Mediates ... · of nuclear receptor corepressor complexes was inhibited by LXR ligand treatment, maintaining the CRP gene in a repressed