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Circulation Research. 2006;99:e88-e99
Published online before print November 16, 2006, doi: 10.1161/01.RES.0000252878.34269.06
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(Circulation Research. 2006;99:e88.)
© 2006 American Heart Association, Inc.

UltraRapid Communication

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

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

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; Howard Hughes Medical Institute, Molecular Biology Institute, and Department of Pathology and Laboratory Medicine (P.T.), University of California, Los Angeles; and Max-Delbrueck Center for Molecular Medicine, Department of Molecular and Clinical Cardiology (F.B.), Franz-Volhard Clinic, HELIOS Clinics GmbH, Charité, Humboldt University, Berlin, Germany.

Correspondence to Rajendra K. Tangirala, PhD, Assistant Professor of Medicine, Division of Endocrinology, Diabetes and Hypertension, David Geffen School of Medicine, University of California, Los Angeles, CA 90095. E-mail rtangirala{at}mednet.ucla.edu


*    Abstract
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 *Abstract
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 down arrowResults
 down arrowDiscussion
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C-reactive protein (CRP), the prototypical human acute phase protein, is an independent risk predictor of future cardiovascular 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 macrophages and attenuates the development of atherosclerosis in various animal models. We demonstrate herein that 2 synthetic LXR ligands, T0901317 and GW3965, inhibit interleukin-1ß/interleukin-6–induced CRP mRNA and protein expression in human hepatocytes. Knockdown of LXR{alpha}/ß by short interfering RNAs completely abolished the inhibitory effect of the LXR agonist T0901317 on cytokine-induced CRP gene transcription. Transient transfection experiments with 5'-deletion CRP promoter constructs identified a region from –125 to –256 relative to the initiation site that mediated the inhibitory effect of LXR ligands on CRP gene transcription. Depletion of the nuclear receptor corepressor by specific short interfering RNA increased cytokine-inducible CRP mRNA expression and promoter activity and reversed LXR ligand–mediated repression of CRP gene transcription. Chromatin immunoprecipitation assays indicated that nuclear receptor corepressor is present on the endogenous CRP promoter under basal conditions. Cytokine-induced clearance of nuclear receptor corepressor complexes was inhibited by LXR ligand treatment, maintaining the CRP gene in a repressed state. Finally, treatment of C57Bl6/J mice with LXR ligands attenuated lipopolysaccharide-induced mouse CRP and serum amyloid P component gene expression in the liver, whereas no effect was observed in LXR{alpha}ß knockout mice. Our observations identify a novel mechanism of inflammatory gene regulation by LXR ligands. Thus, inhibition of CRP expression by LXR agonists may provide a promising approach to impact initiation and progression of atherosclerosis.


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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
There is compelling evidence that inflammatory processes are key mechanisms in all phases of the atherosclerotic process, from lesion initiation to progression and, ultimately, the thrombotic complications of atherosclerosis, such as myocardial infarction and stroke.1,2 Numerous studies have shown that high-sensitivity C-reactive protein (CRP) levels independently predict risk of future cardiovascular events in both healthy individuals and in patients with established coronary artery disease (CAD).3,4 Moreover, the predictive value of high-sensitivity CRP for cardiovascular events in individuals initially free of reported CAD was significantly higher than that associated with other CAD risk markers such as low-density lipoprotein (LDL) cholesterol or lipoprotein (a).5 Beyond the ability of high-sensitivity CRP to predict the risk of future cardiovascular events, previous studies suggest that CRP may have direct proatherogenic properties. For example, CRP was found to upregulate expression of adhesion molecules on endothelial cells6; participate in foam cell formation by mediating LDL uptake by macrophages7 and activate the complement system both in vivo8 and in vitro.9 Furthermore, our group recently demonstrated that CRP induces apoptosis in human vascular smooth muscle cells,10 which may lead to increased plaque thrombogenicity and instability, resulting in an acute cardiovascular event. Although beneficial functions of CRP have been reported, such as upregulation of complement/inhibitory proteins in endothelial cells,11 decrease of P-selectin expression in platelets12 and inhibition of neutrophil migration, the published evidence suggests a proatherogenic effect of CRP. The contradictory effects of CRP may be related to bacterial endotoxin or other contaminants of CRP or may be explained by the existence of 2 distinct conformations of CRP, the native pentamer and a modified, monomeric form.13 Clinical trials are currently underway to determine whether CRP expression may potentially provide a novel pharmacological target for treatment and prevention of atherosclerosis.

CRP is primarily synthesized in hepatocytes, although a growing body of evidence indicates extrahepatic production of CRP, such as in macrophages, adipose tissue or endothelial or smooth muscle cells.14–17 In human hepatoma Hep3B cells, CRP gene expression was found to be modestly induced by interleukin (IL)-6, whereas IL-1ß, which alone has no effect on CRP transcription, synergistically increases IL-6–induced CRP synthesis.18 The transcription factors STAT3 (Signal Transducer and Activator of Transcription 3), nuclear factor {kappa}B and members of the C/EBP family (CCAAT box/Enhancer-Binding Protein) participate in cytokine-induced transcriptional activation of the CRP gene.19,20 In addition, hepatocyte nuclear factor-1 (HNF-1), HNF-3, and Oct-1 also play important roles in the regulation of CRP expression.21–23

The liver X receptors {alpha} (LXR{alpha}) and LXRß (also known as NR1H3 and NR1H2, respectively) are members of the nuclear hormone receptor superfamily and have been suggested as potential targets for therapeutic intervention in human cardiovascular and metabolic disease.24,25 LXR{alpha} is highly expressed in the liver and at lower levels in macrophages, intestine, adipose tissue, lung, and kidney, whereas LXRß is ubiquitously expressed.26 LXR{alpha} and LXRß are ligand-activated transcription factors that form heterodimers with the retinoid X receptor.27 Endogenous activators of LXRs are oxidized cholesterol derivates (oxysterols).28 In addition to endogenous ligands, a number of synthetic LXR agonists, such as T0901317 and GW3965, have been developed. These compounds activate both LXR{alpha} and LXRß.29 Previous studies identified LXRs as important regulators of reverse cholesterol transport and lipid and glucose metabolism.30 In addition LXRs also play a role in innate immunity and regulate inflammatory gene expression in macrophages.31–33 Indeed, synthetic LXR agonists have been shown to both delay the development of atherosclerosis in genetically prone mouse models and induce regression of preexisting atherosclerotic lesions.34–36 In the absence of ligand, heterodimers of LXR and retinoid X receptor, in complex with corepressors such as nuclear receptor corepressor (NCoR) and the related factor SMRT (Silencing Mediator of Retinoic acid and Thyroid hormone receptors), are bound to LXR-responsive elements and inhibit target gene transcription.37 Binding of ligand to LXR causes a conformational change that results in release of corepressors and recruitment of coactivators, leading to induction of target genes. However, the mechanism underlying the repression of inflammatory genes by LXR is poorly understood. Previous studies suggest that indirect mechanisms, such as competition for transcriptional coactivators and antagonism of the nuclear factor {kappa}B signaling pathway, are involved.38

The role of LXR in regulating the expression of acute phase proteins in the liver has not yet been investigated. In the present study, we demonstrate that LXR ligands inhibit cytokine-induced CRP expression in human hepatocytes. This effect is, at least partially, mediated by inhibition of cytokine-induced NCoR clearance from the promoter. These observations define a novel function of LXRs in the control of liver gene expression and support the potential utility of LXR ligands to prevent and treat cardiovascular disease.


*    Materials and Methods
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 *Materials and Methods
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Materials
Primary human hepatocytes (PHHs) and hepatocyte culture medium (HCM BulletKit) were obtained from Cambrex Bioscience. The human hepatoma cell line Hep3B and minimum essential medium (MEM) were purchased from American Type Culture Collection. Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane and ECL Western blotting detection reagents were purchased from Amersham Life Science, and horseradish peroxidase–linked anti-rabbit and anti-mouse antibodies were obtained from Cell Signaling. Recombinant human IL-1ß 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 (GlaxoSmithKline Inc, Durham, NC). Antibodies were commercially obtained from the following suppliers: CRP (clone CRP-8) and anti–FLAG M2 from Sigma-Aldrich; NCoR from Affinity BioReagents.

Cell Culture
Human hepatoma Hep3B cells were cultured in MEM supplemented with 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 the instructions of the manufacturer. For ligand treatment, Hep3B cells were serum-deprived in 0% FBS MEM and treated with T0901317 or GW3965 for 18 hours before stimulation with IL-1ß and IL-6, at a final concentration of 20 ng/mL and 10 ng/mL, respectively. PHHs were treated with LXR ligands in HCM supplemented with growth factors for 18 hours before stimulation with IL-6 (10 ng/mL). For all data shown, individual experiments were repeated at least 3 times with different lots of cells.

RNA Isolation and Northern Blotting
Total RNA isolation and Northern blotting were performed as previously described.39 Human CRP cDNA was obtained from Dr Alok Agrawal (East Tennessee State University, Johnson City). Blots were cohybridized with human GAPDH cDNA (Sigma-Aldrich) to assess equal loading of samples.

Western Blot Analysis
Cells were harvested at the indicated time points and sonicated in solubilization buffer (Cell Signaling). Supernatants of Hep3B cells were collected as indicated and concentrated using centrifugal filter units (Millipore). Nuclear extracts were isolated using the Nuclear Extraction Kit (Panomics) according to the instructions of the manufacturer. Western blot analyses were performed as previously described.39

Reverse Transcription and Quantitative Real-Time Polymerase Chain Reaction
Total RNA was isolated using the RNeasy MINI Kit (QIAGEN). Total RNA (400 ng) was reverse transcribed with random hexamers using the TaqMan Reverse Transcription Reagent Kit (Applied Biosystems) according to the instructions of the manufacturer. Real-time quantitative polymerase chain reaction (PCR) assays were performed by using an ABI-PRISM 7700 system (Applied Biosystems) in a total volume of 25 µL. Each sample was analyzed in triplicate, and cycle thresholds of individual genes were normalized to corresponding GAPDH mRNA expression values. Primer and probes used in the experiments were obtained from Applied Biosystems.

Immunofluorescence Microscopy
Hep3B 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-1ß/IL-6 for an additional 24 hours. After 12 hours of cytokine stimulation, BD GolgiStop (containing monensin; BD Biosciences) was added to block the intracellular protein transport processes. Cells were fixed for 15 minutes with 3.7% paraformaldehyde (in PBS) and permeabilized in 0.2% Triton X-100 (in PBS) for 5 minutes. After blocking of nonspecific staining with the Image-iT FX signal enhancer (Molecular Probes), cells were stained overnight with a CRP antibody, followed by a secondary goat anti-mouse IgG (1:200; Molecular Probes). Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI). Cells were visualized with a fluorescence microscope, and colocalization of fluorescein and DAPI staining was performed by overlay projections.

Transient Transfection and Luciferase Assay
Transient transfections were performed in triplicate in 6-well plates, using the FuGENE 6 reagent (Roche) according to the instructions of the manufacturer. Twenty-four hours after transfection, cells were incubated 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-1ß (20 ng/mL) and IL-6 (10 ng/mL) treatment using a Dual Luciferase Reporter Assay System (Promega) according to the instructions of the manufacturer. All experiments were repeated at least 3 times with different cell preparations. The CRP promoter constructs were generously provided 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 Israel Deaconess Medical Center and Harvard Medical School, Boston, Mass).

Short Interfering RNA
Short interfering RNA (siRNA) specific for NCoR, LXR{alpha}, and LXRß (SMARTpool short interfering RNA [siRNA]) and nonsilencing control siRNA were purchased from Dharmacon. Transfections were performed with siRNAs (40 nmol/L) using the transfection reagent DharmaFECT Reagent 4. Forty-eight hours after transfection, cells were serum deprived in the presence of T0901317 or vehicle (DMSO), followed by IL-1ß/IL-6 stimulation as indicated.

Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation (ChIP) assays were performed using a ChIP assay kit from Upstate according to the instructions of the manufacturer. Briefly, cells were pretreated with 5 µmol/L T0901317 for 18 hours and stimulated with a combination of IL-1ß (20 ng/mL) and IL-6 (10 ng/mL) as indicated. Cells were crosslinked with 2 mmol/L disuccinimidyl glutarate for 45 minutes before crosslinking for 15 minutes with 1% formaldehyde. This 2-step crosslinking method has been shown to be more efficient than the conventional single formaldehyde crosslinking procedure.40 After lysis, cells were sonicated using a Branson Digital Sonifier model 450 (Branson Ultrasonics Corporation). Chromatin fragments were immunoprecipitated with an antibody directed against NCoR (2 µg) overnight at 4°C. Rabbit IgG (Santa Cruz) was used as a negative control. DNA fragments were purified from chromatin using the QUIAquick PCR Purification Kit (QIAGEN) according to the instructions of the manufacturer. The final DNA extractions were amplified by PCR, using the following primer pairs: forward, 5'-CAAAGTGGAGCCCTGAGAGA-3'; reverse, 5'-CTACCTC-CTCCTGCCTGGAT-3'. As control, primers against an unrelated region 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 were resolved on 2% agarose gels and visualized using ethidium bromide.

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

Animal Studies
Male C57Bl6/J mice (The Jackson Laboratory) and LXR{alpha}ß-knockout mice on a mixed background (C57Bl6/J and 129Sv), received daily intraperitoneal injections of T0901317 (20 mg/kg body weight per day in DMSO) or vehicle (DMSO). At day 4 of treatment, bacterial LPS (3 mg/kg body weight) was injected intraperitoneally. Twenty-four hours later, animals were euthanized and liver samples were harvested. Serum samples were collected from the mice 24 hours after injection of LPS. All animal protocols were approved by the University of California, Los Angeles Animal Research Committee and complied with all federal, state, and industrial regulations.

Statistical Analysis
ANOVA and paired or unpaired t test were performed for statistical analysis as appropriate. Probability values less than 0.05 were considered to be statistically significant. Results are expressed as mean±SEM.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
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LXR Agonists Suppress CRP Expression in Hep3B Cells
Based on previous studies, we used the human hepatoma cell line Hep3B as a cell culture model to study CRP gene regulation in vitro.41,42 Given their antiatherogenic and antiinflammatory properties, LXR agonists have been proposed as promising therapeutic approach to treat cardiovascular disease.38 To determine the effect of synthetic LXR ligands on cytokine-induced CRP expression, quiescent Hep3B cells were pretreated with T0901317 or GW3965 for 18 hours and stimulated with a combination of IL-1ß (20 ng/mL) and IL-6 (10 ng/mL) for an additional 24 hours. As shown in Figure 1A, both synthetic LXR ligands inhibited IL-1ß/IL-6–stimulated CRP mRNA expression in a dose-dependent manner, as determined by Northern blotting and quantitative real-time RT-PCR (76.1% inhibition at 5 µmol/L T0901317 and 69.1% inhibition at 5 µmol/L GW3965 versus IL-1ß/IL-6 alone; P<0.05). LXR agonist–mediated inhibition of CRP mRNA expression also correlated with reduced levels of CRP protein expression, as analyzed by immunofluorescence staining, Western blot analyses, and ELISA. As shown in Figure 1B, no cellular CRP protein expression could be detected in quiescent Hep3B cells by immunofluorescence, whereas stimulation with IL-1ß (20 ng/mL)/IL-6 (10 ng/mL) resulted in profound cytoplasmic staining. Pretreatment with two different LXR ligands, T0901317 and GW3965 (both at 5 µmol/L), inhibited cytokine-induced CRP protein expression. Similarly, cytokine-induced CRP protein release to the culture supernatant was suppressed by LXR ligands in a dose-dependent manner (Figure 1C). Together, these data demonstrate that LXR ligands inhibit cytokine-induced CRP mRNA and protein expression in Hep3B cells.


Figure 1
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  		Figure 1. The synthetic LXR agonists T0901317 and GW3965 inhibit cytokine-induced CRP mRNA and protein expression in Hep3B cells. Hep3B cells were pretreated in MEM medium containing 0% FBS for 18 hours with vehicle (DMSO) or the indicated concentrations of LXR ligands. Cells were then stimulated with a combination of IL-1ß (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 normalized to GAPDH mRNA expression. Data are presented as mRNA levels relative to untreated control (Con). B, CRP protein expression was 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 assayed by Western blotting using a CRP-specific antibody and ELISA. Quantification was performed from 3 independently performed experiments. Results are presented as mean±SEM. *P<0.05 vs IL-1ß/IL-6–stimulated cells.

LXR Ligands Inhibit CRP Expression in PHHs
We next investigated whether the inhibitory effect of LXR agonists on cytokine-induced CRP expression is also observed in PHHs. Cells were stimulated with 10 ng/mL IL-6 alone, because, in contrast to Hep3B cells, IL-6–stimulated CRP expression is reduced by costimulation with IL-1ß.43 As depicted in Figure 2, preincubation of PHHs with either T0901317 or GW3965 resulted in a significant suppression of IL-6–induced (10 ng/mL) CRP mRNA expression.


Figure 2
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  		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 were then harvested and analyzed for CRP mRNA expression by quantitative real-time PCR. Results were normalized to corresponding GAPDH mRNA expression values. Data are expressed as fold induction±SEM over unstimulated control (Con). *P<0.05 vs IL-6–treated cells.

LXR Activation Suppresses Cytokine-Induced CRP Transcription
The mechanism for the inhibition of cytokine-induced CRP transcription by LXR ligands is unlikely to be direct, as activation of LXR by its ligands has not been reported to function as a transcriptional repressor. Moreover, sequence analysis of the 5'-flanking region of the CRP promoter did not reveal the presence of potential LXR response elements. To identify the promoter elements that mediate the transcriptional suppression of CRP in Hep3B cells by LXR ligands, we used a series of 5'-deletion constructs (Figure 3A). We found, as previously reported, that the effect of IL-1ß/IL-6 stimulation was significantly lower in deletion –125CRP-Luc as compared with longer promoter constructs and that inducibility by cytokines was lost in the –55CRP-Luc and –86CRP-Luc constructs.41 Moreover, although both the –256CRP-Luc and –125CRP-Luc constructs retained cytokine inducibility, only the –256CRP-Luc construct demonstrated transcriptional repression by the LXR ligand T0901317. These findings indicate that the ability of LXR to inhibit the CRP promoter is dependent on transcription factor binding sites located between –256 and –125 relative to the transcription initiation site. We also performed promoter assays using various concentrations of LXR ligand. As demonstrated in Figure 3B, T0901317 dose-dependently attentuated IL-1ß/IL-6–induced CRP promoter activity over a concentration range of 1 to 5 µmol/L.


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

LXR Ligand-Mediated Inhibition of CRP Expression Is Receptor Dependent
To address whether the inhibitory effect observed with T0901317 is mediated by LXRs, we used siRNAs to knockdown LXR{alpha} and LXRß. Inhibition of LXR{alpha}/ß expression in Hep3B cells using specific siRNAs completely abolished the ability of T0901317 (2 µmol/L) to inhibit cytokine-induced CRP mRNA expression (Figure 4A) and promoter activity (Figure 4B). In addition, cytokine-induced CRP gene transcription and promoter activity was enhanced in Hep3B cells transfected with the specific siRNA compared with control siRNA transfected cells. Taken together, these data demonstrate that LXR ligands repress CRP through a receptor dependent mechanism.


Figure 4
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  		Figure 4. LXR ligands mediate CRP gene repression through a receptor-dependent mechanism. A, Hep3B cells were transfected either with scrambled siRNA or LXR{alpha}/ß siRNA. Forty-eight hours after transfection, cells were serum deprived in the presence of vehicle (DMSO) or the LXR agonist T0901317. Cells were stimulated with IL-1ß (20 ng/mL)/IL-6 (10 ng/mL) for 24 hours, and total RNA was analyzed for CRP mRNA expression by quantitative real-time PCR. B, Hep3B cells were transiently transfected with the Luc-904 CRP promoter construct and the indicated siRNAs. Following transfection, cells were treated as described in A and luciferase activities were analyzed. Experiments were performed in triplicate and data are presented as mean±SEM. *P<0.05 vs vehicle, #P<0.05 vs IL-1ß/IL-6–stimulated cells.

Inhibition of CRP Transcription Activity by NCoR
Recent studies indicate that NCoR complexes are involved in basal suppression of inflammatory response genes in macrophages, with loss of NCoR resulting in derepression of nuclear factor {kappa}B and activator protein-1 target genes.44,45 This interaction between NCoR and inflammatory genes prompted us to address the role of NCoR in the regulation of cytokine-induced CRP transcription in Hep3B cells. Therefore, we transiently transfected the –904CRP-Luc promoter construct along with an expression vector for NCoR. Nuclear localization of overexpressed NCoR was confirmed by indirect immunofluorescence (Figure 5A). In accordance with previous observations,46 NCoR-transfected cells displayed a characteristic nuclear speckle staining (Figure 5A), whereas an IgG control antibody exhibited only background staining (data not shown). As shown in Figure 5B, cotransfection of the NCoR expression vector resulted in a dose-dependent inhibition of IL-1ß/IL-6–induced CRP promoter activity, indicating that NCoR participates in regulation of CRP transcriptional activity.


Figure 5
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  		Figure 5. Overexpression of NCoR inhibits cytokine-induced CRP promoter activity. Hep3B cells were cotransfected with a FLAG-tagged full-length NCoR expression vector and the Luc-904 CRP promoter construct as indicated. A, Using a monoclonal anti–FLAG M2 antibody, followed by a fluorescein-conjugated (green) secondary antibody, NCoR protein was detected in the nucleus, but not cytoplasm of transfected cells. The cell nuclei were stained with DAPI (blue). B, After transfection, cells were incubated in 0% FBS MEM medium for 18 hours, followed by stimulation with a combination of IL-1ß (20 ng/mL) and IL-6 (10 ng/mL) for 24 hours. Luciferase activities were assayed as described in Figure 4. Results are expressed as fold induction vs untreated control and presented as mean±SEM. *P<0.05 vs vehicle.

NCoR-Specific siRNA Reverses LXR-Mediated Inhibition of CRP Transcription
We next used a NCoR-specific siRNA to evaluate whether inhibition of NCoR expression results in reversal of repression of CRP gene transcription by LXR ligands. Efficacy of NCoR knockdown by siRNA was evaluated by Western blot analyses of NCoR protein expression in Hep3B cells transfected with the specific siRNA and a control siRNA (Figure 6A). Inhibition of NCoR expression using a NCoR-specific siRNA resulted in attenuation of LXR ligand–mediated inhibition of CRP mRNA transcription compared with control siRNA-transfected cells (53.1% inhibition versus 80.2% inhibition at 5 µmol/L T0901317; P<0.05; Figure 6B). Consistent with these findings, knockdown of NCoR expression reversed LXR ligand–mediated CRP promoter repression (47.6% inhibition versus 77.5% inhibition at 5 µmol/L T0901317; P<0.05; Figure 6C). Taken together, these data indicate that inhibition of CRP gene transcription is mediated, at least in part, through a NCoR dependent mechanism.


Figure 6
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  		Figure 6. siRNA targeting NCoR reverses LXR ligand–mediated repression of CRP gene transcription. A, Efficacy of NCoR siRNA was evaluated by transfecting Hep3B cells with the indicated NCoR-specific and control siRNA as described in Material and Methods. Nuclear protein levels were determined by Western blot analyses using a NCoR-specific antibody. B, Hep3B cells were transfected with control siRNA or a NCoR-specific siRNA. Following transfection, cells were pretreated for 18 hours with vehicle (DMSO) or LXR ligand before stimulation with IL-1ß (20 ng/mL)/IL-6 (10 ng/mL) for 24 hours. Total RNA was isolated, and the level of CRP mRNA was examined by quantitative real-time PCR. C, Cells were transiently transfected with the Luc-904 CRP promoter construct and the indicated siRNAs as described in Material and Methods. Forty-eight hours after transfection, cells were treated as described in B and luciferase activity was measured. Experiments were performed in triplicate; data are expressed as fold induction vs control and presented as mean±SEM. *P<0.05 vs vehicle, #P<0.05 vs IL-1ß/IL-6–stimulated cells.

LXR Activation Prevents Cytokine-Induced Dissociation of NCoR From the CRP Promoter
Our previous observations predicted that NCoR complexes associate with the CRP promoter. To confirm that NCoR binds to the endogenous CRP promoter and that LXR ligands interfere with this binding in vivo, we next performed ChIP experiments. PCR amplification using primer pairs that cover a region from –6 to –241 in the CRP promoter demonstrated that NCoR is present on this CRP promoter sequence under basal conditions (Figure 7). Time-course experiments revealed that NCoR was cleared from the CRP promoter within 60 minutes of IL-1ß/IL-6 treatment. However, clearance of NCoR was substantially inhibited by T090137 treatment. ChIP analysis with primers against an unrelated CRP promoter region revealed no NCoR binding. Together, these data demonstrate that LXR ligands prevent cytokine-induced dissociation of NCoR from the CRP promoter, thus maintaining the CRP gene in a repressed state.


Figure 7
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  		Figure 7. LXR ligand prevents cytokine-induced dissociation of NCoR from the CRP promoter. ChIP assays were performed using chromatin isolated from serum-deprived Hep3B cells pretreated for 18 hours with vehicle (DMSO) or T0901317 (5 µmol/L), followed by stimulation with IL-1ß (20 ng/mL)/IL-6 (10 ng/mL). Crosslinked cell lysates were subjected to immunoprecipitation with rabbit IgG as control (lane 1) or a NCoR-specific antibody (lanes 2 to 6). DNA precipitates were isolated and then subjected to PCR using primers corresponding to the region from –241 to –6 of the CRP gene (a). Input DNA was detected by using 10% of the soluble chromatin before immunoprecipitation. PCR with primers against an unrelated promoter region upstream (b) and downstream (c) from –241 to –6 revealed no NCoR binding. The data shown is representative of 3 independently performed experiments.

LXR Agonists Inhibit Acute Phase Response Gene Expression in Vivo
Although CRP is the major acute phase protein in humans and most mammals, serum amyloid P component (SAP) is the major acute-phase protein in the mouse.47 To finally determine whether the effects of LXR ligands to suppress acute phase response gene expression are applicable in vivo, the synthetic LXR agonist T0901317 (20 mg/kg body weight per day) or vehicle (DMSO) was administered to C57Bl6/J and LXR{alpha}ß knockout mice 3 days before intraperitoneal LPS injection (3 mg/kg body weight). Twenty-four hours after LPS treatment animals were euthanized. Hepatic mRNA expression levels of SAP and CRP in LXR ligand–treated C57Bl6/J mice were significantly decreased compared with those in vehicle-treated mice (60.2% and 76.9% inhibition versus LPS alone; n=4, P<0.05; Figure 8). At 24 hours after injection of LPS, serum levels of SAP, as determined by ELISA, were significantly reduced in C57Bl6/J mice treated with T0901317 (38.6% inhibition versus LPS alone; n=4, P<0.05; Figure 8B). The ability of the LXR agonist T0901317 to inhibit LPS-induced acute phase response gene expression is strictly receptor-dependent, because no effect on CRP and SAP mRNA (Figure 8A) and SAP serum levels (Figure 8B) was observed in ligand-treated LXR{alpha}ß–/– mice. Taken together, these results show that LXR agonists inhibit acute-phase response gene expression in vivo and further emphasize the important role of LXRs as modulators of inflammatory gene response both in vitro and in vivo.


Figure 8
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  		Figure 8. The LXR agonist T0901317 inhibits LPS-induced APR gene expression in the liver in vivo. C57Bl6/J and LXR{alpha}ß knockout (DKO) mice were pretreated with T0901317 (20 mg/kg per day) or vehicle (DMSO) for 4 days, followed by intraperitoneal injection of saline or LPS (3 mg/kg body weight), respectively. A, Mouse CRP and SAP mRNA expression in the liver were analyzed by quantitative real-time PCR. Relative mRNA expression levels were determined after normalization to internal control GAPDH mRNA levels. B, Serum levels of SAP in the mice were determined at 24 hours after injection of saline or LPS. Results are presented as mean±SEM. *P<0.05 vs vehicle treated mice, #P<0.05 vs LPS injected mice (n=4 mice per group).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
LXR ligands play a dual role in maintaining lipid and lipoprotein homeostasis and in modulating inflammatory gene expression in both macrophages and lymphocytes.32,48,49 In animal models of atherosclerosis, such as apolipoprotein E–deficient (apoE–/–) and LDL receptor–deficient (LDLR–/–) mice, we found that LXR ligand treatment reduced the development of atherosclerosis.34 Moreover, recent studies revealed that the LXR signaling pathway links innate immunity to macrophage cholesterol metabolism.50 Thus, the ability of LXRs to promote reverse cholesterol transport, attenuate inflammation, and improve glucose tolerance identifies the LXR pathway as a potential target for novel therapeutic interventions in human cardiovascular disease.38,51,52 In the present study, we demonstrate that LXR ligands attenuate cytokine-induced CRP expression in both Hep3B cells and PHHs. This effect is, at least partially, mediated by inhibition of NCoR clearance from the CRP promoter, which prevents the switch from active repression to transcriptional activation. The observations described herein identify a novel mechanism by which LXR agonists inhibit inflammatory gene responses and expand the role for LXR agonists as antiinflammatory ligands to hepatocytes.

The human hepatoma cell line Hep3B is a widely used model for studying the regulation of CRP gene transcription. The major inducer of CRP gene transcription in Hep3B cells is IL-6; however, maximal gene expression is achieved only when both IL-1ß and IL-6 are present.41 The results presented here demonstrate that LXR ligands are negative regulators of CRP gene expression in hepatocytes. In both Hep3B cells and primary hepatocytes, cytokine-induced CRP mRNA and protein expression was significantly inhibited by the 2 synthetic LXR agonists, T0901317 and GW3965. siRNA-mediated depletion of LXR{alpha}/ß abolished the inhibitory effect of T0901317 on CRP gene expression, demonstrating a receptor-dependent mechanism. However, analysis of the CRP promoter did not reveal the presence of any putative LXR-responsive elements, indicating that the inhibition of cytokine-induced CRP transcription by LXR ligands is indirect through inhibition of binding of other transcription factors, competition for transcriptional activators, or recruitment of corepressor complexes. Previous studies have highlighted important roles for various transcription factors, such as STAT3, C/EBP-ß/{delta}, HNF-1{alpha}, HNF-3, or Oct-1 in regulating CRP gene expression.19,21–23 To localize the regions important for LXR-dependent repression, we used a series of 5'-deletion constructs. We identified a region in the CRP promoter between –256 and –125 that is essential for LXR ligand–mediated inhibition of cytokine-induced transcriptional activity. This promoter region contains a binding site for HNF-1{alpha}. Previous studies have shown that mutations in either of these 2 HNF-1 sites abolished CRP gene induction.21 In addition, HNF-1{alpha} overexpression induced CRP promoter activity in Hep3B cells in the absence of IL-1ß/IL-6, whereas transfection with a dominant negative HNF-1{alpha} expression vector had no effect (data not shown). However, despite the important role of the HNF-1{alpha} site for the transcriptional regulation of the CRP gene, electrophoretic mobility-shift assays revealed no significant changes in protein/DNA complexes in the presence of LXR ligands (data not shown).

This observation prompted us to investigate the role of corepressors in LXR ligand–mediated inhibition of CRP gene transcription. Previous studies demonstrate that NCoR exerts repressive effects on basal inflammatory gene expression by interacting with several classes of DNA-binding transcription factors,53–55 with loss of NCoR resulting in gene transcription in macrophages.45 Recently, Pascual et al defined a novel pathway by which peroxisome proliferator-activated receptor {gamma} (PPAR-{gamma}) represses inflammatory gene responses in macrophages.44 PPAR-{gamma} ligand–induced SUMOylation targets PPAR-{gamma} to NCoR-containing complexes associated with inflammatory gene promoters in the basal state. This in turn prevents LPS-mediated clearance of NCoR complexes from the promoter by inhibiting the recruitment of the ubiquitylation/19S proteosome machinery. We thus explored the possibility that the inhibitory effect on CRP expression observed with LXR ligands is mediated by NCoR. Overexpression of NCoR resulted in a potent inhibition of cytokine-induced CRP promoter activity, suggesting that NCoR plays an important role in regulating CRP gene transcription. Consistent with these findings, knockdown of NCoR using a NCoR-specific siRNA further increased cytokine-induced CRP gene transcription. Furthermore, inhibition of NCoR expression by siRNA reversed LXR ligand–dependent inhibition of CRP mRNA expression and promoter activity. These findings suggest that NCoR complexes associate with the CRP promoter in the basal state. ChIP assays revealed that NCoR was bound to the CRP promoter under basal conditions. Moreover, cytokine stimulation cleared NCoR from the promoter, which was inhibited by treatment of cells with the LXR ligand. These studies suggest that the presence of NCoR complexes on the promoter maintains the CRP gene in a repressed state, preventing the switch from active repression to transcriptional activation. Additional studies are necessary to elucidate in more detail the LXR ligand–mediated inhibition of NCoR clearance from the CRP promoter and to identify the transcription factors interacting with the NCoR corepressor complex.

We further addressed whether LXR{alpha} or LXRß expression in hepatocytes is regulated in response to LXR ligands. LXR{alpha} is highly expressed in the liver, whereas LXRß is ubiquitously expressed.26 Whereas treatment of Hep3B cells with LXR ligands had no effect on LXRß mRNA expression, LXR agonists dose-dependently induced LXR{alpha} mRNA (Figure I in the online data supplement). Similar to the effect on LXR{alpha} expression, the LXR target gene ABCA1 was upregulated by LXR agonists (data not shown). The observed autoregulation of the LXR{alpha} gene in liver might have implications for the inhibitory effect of LXR ligands on CRP gene expression. However, previous studies performed in macrophages obtained from LXR{alpha}–/– and LXRß–/– mice indicate that both LXR isoforms are negative regulators of inflammatory gene expression.48 The first direct target gene of LXRs to be identified in mice was Cyp7a1, the rate-limiting enzyme in hepatic bile acid synthesis.56 Subsequent studies demonstrated that activation of hepatic LXR also exerts potentially beneficial effects by regulating genes involved in cholesterol metabolism. Induction of ABCG5 and ABCG8, members of the ATP-binding cassette (ABC) superfamily, results in increased 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 antiatherogenic effects of LXR activation.58 However, in addition to modulating cholesterol metabolism, LXRs also induce lipogenic genes. Mice treated with synthetic LXR agonists upregulate fatty acid synthase, acetyl-coenzyme A carboxylase and the transcription factor SREBP-1c, resulting in an increase of hepatic and plasma triglycerides.38 The development of gene- or isoform-specific LXR agonists is a promising approach to induce the desired beneficial effects of LXR activation without the detrimental hepatic effects.

Numerous studies indicate that CRP, in addition to being a risk marker for future cardiovascular events, plays a direct role in the development and progression of atherosclerotic lesions.59 Thus, approaches to directly target the synthesis of CRP or blocking the function of CRP by inhibitors might be a promising new tool for both primary prevention and treatment of cardiovascular disease. Although extrahepatic synthesis of CRP, such as by macrophages, neuronal cells, adipose tissue, and renal cortical tubular epithelial cells has been shown,15,60–62 the principal source of circulating CRP is the hepatocyte, which synthesizes CRP under the transcriptional control of inflammatory cytokines, in particular IL-6.63 In mice, SAP and serum amyloid A are the major acute-phase response (APR) genes, whereas CRP, because of its modest regulation, is considered a minor APR gene. However, several common molecular properties of SAP and CRP have been established. Both are members of the pentraxin family, with a similar pentagonal arrangement of their subunits and display calcium-dependent binding to specific substrates.64,65 In addition, both mouse SAP and human CRP respond to IL-1ß and IL-6 stimulation, which enhances binding of STAT3 and C/EBPß to their promoters. In contrast, previous findings indicate that only STAT3 is involved in mouse CRP gene expression rather than a complex, synergistic interaction between STAT3 with C/EBP-ß.66 These differences in the mouse CRP and SAP promoter regions might account for the different transcriptional response during inflammation. We found a significant increase in liver CRP and SAP mRNA levels 24 hours after LPS injection of C57Bl6/J and LXR{alpha}ß–/– mice. In C57Bl6/J mice, induction of these APR genes was significantly inhibited by pretreatment with the LXR ligand T0901317. In contrast, the ability of T0901317 to inhibit LPS-induced expression of CRP and SAP was completely abolished in LXR{alpha}ß–/– mice, indicating a receptor-dependent mechanism. Thus, our results extend the inhibitory effect of LXR ligands on human CRP gene transcription in vitro to related APR genes in vivo.

In summary, results presented in this study demonstrate that LXR agonists inhibit cytokine-induced CRP expression in hepatocytes and negatively interfere with cytokine-induced NCoR dissociation from the CRP promoter, thus maintaining the CRP gene in a repressed state. LXR ligand–mediated attenuation of CRP expression in vitro translates into in vivo as LPS-induced APR gene expression in murine liver is inhibited in LXR agonist–treated animals. Thus, inhibition of CRP expression by LXR agonists may offer a novel therapeutic approach for the primary prevention and treatment of atherosclerotic disease.


*    Acknowledgments
 
We thank Dr Alok Agrawal and Dr David Samols for providing the CRP cDNA and promoter constructs and Dr Anthony Hollenberg for providing the NCoR expression vector. We thank Rima Boyadjian for her excellent technical assistance with the ELISA assays.

Sources of Funding

This study was supported by NIH grant HL075171 to (W.A.H.). F.B. was supported in part by a research fellowship from Philip Morris USA Inc. Y.T. was supported by Japan Heart Foundation/Bayer Yakuhin Research Grant Abroad. P.T. is an investigator of the Howard Hughes Medical Institute.

Disclosures

None.


*    Footnotes
 
Original received July 17, 2006; revision received October 30, 2006; accepted November 8, 2006.


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