| BackgroundCoronary atherosclerosis represents the leading cause of morbidity and mortality of men and women throughout the western world. Hypercholesterolemia is a well-established risk factor for the incidence of atherosclerosis and its pathological complications. Evidence from clinical trials indicates that reducing plasma cholesterol by dietary and/or pharmacological means leads to reductions in the incidence of death from cardiovascular disease. Appealing therapeutic targets for reducing hypercholesterolemia and atherosclerosis include members of the nuclear hormone receptor superfamily, such as the peroxisome proliferator-activated receptor and the liver X receptor subfamilies, among others. These ligand-activated transcription factors regulate various metabolic processes by controlling the expression of specific gene cassettes.In addition to these well-characterized ligand-activated transcription factors, the nuclear receptor (NR) superfamily comprises many orphan receptors, whose ligands and physiological functions remain unknown. Among this group of orphan receptors is the NR4A subfamily, including Nur77(NR4A1), Nurrl (NR4A2), and Nor-1(NR4A3). In contrast to other members of the superfamily, NR4A nuclear receptors are’immediate early genes’, and are transiently and rapidly induced by a pleiotropy of environmental cues. Nur77is a member of the NR4A subfamily and consists, like other nuclear receptors, of an N-terminal activating function-1(AF-1) domain, a central two zinc-finger DNA-binding domain (DBD), and a C-terminal ligand binding domain (LBD). Early functional studies have pointed to a critical role of Nur77in regulating differentiation, proliferation, and apoptosis. More recent research has characterized Nur77as key transcriptional regulators of glucose and lipid homeostasis, adipogenesis, inflammation, and vascular remodeling. Initial experiments have demonstrated that Nur77promotes lipolysis in muscle. Subsequently, studies reveal that Nur77modulates plasma lipoprotein profiles and hepatic lipid metabolism in mice. Consistent with these data, the recent report have noted that the hepatic steatosis and increased transcription factors sterol regulatory element-binding binding protein lc (SREBPlc) expression can be observed in Nur77-deficient mice fed a high-fat diet. The present study evaluated the effect of Nur77on cholesterol metabolism in THP-1macrophage-derived foam cells, and on plasma lipoprotein profiles, circulating cytokine levels, hepatic lipid deposition and the development of aortic atherosclerosis in apoE-/-mice.Aims1. To study the effect of Nur77on lipid loading, lipid content and cholesterol efflux.2. To study the effect of Nur77on plasma lipid parameters and circulating cytokinelevels in apoE-/-Mice.3. To study the effect of Nur77on hepatic lipid deposition in apoE-/-mice.4. To study the effect of Nur77on gene expression involved in intestinal lipid absorption.5. To study the effect of Nur77on plaque formation in ApoE-/-Mice.Methods1. Effect of Nur77on lipid loading, lipid content and cholesterol efflux by in THP-1macrophage-derived foam cells by treatment with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmid over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77).1.1Human monocytic THP-1cells were maintained in RPMI1640medium containing10%fetal calf serum (FCS) in the presence of streptomycin (100μg/mL), penicillin (100U/ml) and differentiated for72h with100nM phorbol12-myristate13-acetate (PMA). Macrophages were transformed into foam cells by incubation in the presence or absence of50mg/ml ox-LDL in serum-free RPMI1640medium containing0.3%bovine serum albumin (BSA) for48h. Cells were seeded in6-or12-well plates or60-mm dishes and grown to80-90%confluence before use.1.2The PCR-XL-TOPO vector containing Nur77, vector PIRES2-EGFP, Platinum HIFI Taq polymerase and Accuprime Pfx DNA polymerase were purchased from Invitrogen Biotechnology (Shanghai, China). The pCDNA3.1(+) vector, competent DH5a cells, Xhol and EcoRI restriction enzymes and T4DNA ligase were purchased from TaKaRa Biotechnology Co. Ltd.(Dalian, China). The fragment of EcoRI-Nur77-IRES-EGFP-Xhol was achieved from the PCR-XL-TOPO vector and PIRES2-EGFP vector by overlap extension PCR and was linked to pcDNA3.1to create the recombinant plasmid pcDNA3.1-Nur77-IRES-EGFP. The inserted gene was identified by electrophoresis and sequencing. The recombinant plasmid was then transfected into the cultured cells by Lipofectamine2000(Invitrogen), and the over-expression effects of Nur77was confirmed by RT-PCR and western blotting.1.3Short-interfering RNA (siRNA) specific for human Nur77and nonsilencing control siRNA were synthesized by Guangzhou RiboBio, China. Cells (2×106/well) were transfected using Lipofectamine2000. Forty-eight h post-transfection, real-time RT-PCR and Western blotting were performed. Based on the Western blot analysis, the Nur77siRNA suppressed the expression of Nur77proteins by83%as compared to the control siRNA in THP-1macrophage-derived foam cells.1.4Total RNA from cultured cells was extracted using TRIzol reagent (Invitrogen) in accordance with the manufacturer’s instructions. Real-time quantitative PCR, using SYBR Green detection chemistry, was performed on the ABI7500Fast Real Time PCR system (Applied Biosystems, Foster City, CA, USA). Melt curve analyses of all real-time PCR products were performed and shown to produce a single DNA duplex. All samples were measured in triplicate and the mean value was considered for comparative analysis. Quantitative measurements were determined using the AACt method and GAPDH expression was used as the internal control.1.5PMA-differentiated THP-1cells were treated with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmids over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77) as indicated, then fluorescent-tagged Dil-oxLDL was added, and the cells were incubated for24h. Adherent cells were harvested, washed three times with phosphate buffer saline (PBS). Analysis was performed on a fluorescent activated cell sorting (FACS) calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with Cell Quest Pro software (BD Biosciences, San Jose, CA, USA).1.6The sterol analyses were performed using a HPLC system (model2790, controlled with Empower Pro software; Waters Corp., Milford, MA, USA). Absorbance at216nm was monitored. Data were analyzed with TotalChrom software from PerkinElmer (Waltham, MA, USA).1.7Cells were cultured and treated with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmids over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77), as indicated above. Then, they were labeled with0.2μCi/ml [3H]cholesterol. After72h, cells were washed with PBS and incubated overnight in RPMI1640medium containing0.1%(w/v) BSA to allow equilibration of [3H]cholesterol in all cellular pools. Equilibrated [3H]cholesterol-labeled cells were washed with PBS and incubated in2ml of efflux medium containing RPMI1640medium and0.1%BSA with25μg/ml human plasma apoA-I. A150μl sample of efflux medium was obtained at the times designated and passed through a0.45-μm filter to remove any floating cells. Monolayers were washed twice with PBS, and cellular lipids were extracted with isopropanol. Medium and cell-associated [3H]cholesterol was then measured by liquid scintillation counting. Percent efflux was calculated by the following equation:[total media counts/(total cellular counts+total media counts)]×100%. 2. Effect of Nur77on plasma lipid parameters and circulating cytokine levels in apoE-/-mice by treatment with Nur77agonist, lentivirus encoding mouse Nur77(Ad-Nur77) and lentivirus encoding mouse Nur77(si-Nur77).2.1The serum concentrations of IL-1β, IL-6and TNF-a were measured in duplicate using a commercial ELISA kit (R&D Systems, Minneapolis, MN, USA). Serum CRP amount was measured in duplicate using a commercial ELISA kit (Diagnostic System Laboratories, Webster, TX, USA).2.2The serum apolipoprotein A1(apoA1) and apoB100concentrations were measured in duplicate using a commercial ELISA kit (Cusabio Biotech Co., Ltd., China). The T-Cho, TG, LDL-C, HDL-C and VLDL-C concentrations were determined enzymatically using an automated analyzer.3. Effect of Nur77on hepatic lipid deposition in apoE-/-mice by treatment with Nur77agonist, lentivirus encoding mouse Nur77(Ad-Nur77) and lentivirus encoding mouse Nur77(si-Nur77).3.1Hepatic lipid deposition was assessed in samples embedded in OCT compound by Oil Red O staining. Briefly, liver cryosections were fixed for10min in60%isopropanol and stained with0.3%Oil Red O in60%isopropanol for30min and subsequently washed with60%isopropanol. Sections were counterstained with Gill’s hematoxylin, washed with acetic acid solution (4%), and mounted with aqueous solution. Once stained, sections were quantified by histomorphometry.3.2For the procedure, each frozen liver tissue was sectioned at5μM thicknesses and fixed to microscope slides. Sections of frozen tissue specimens were mounted on polyl-lysine (Sigma, St. Louis, MO)-coated slides, air dried, and fixed with acetone. Immunohistochemical staining was performed for Nur77using Rabbit polyclonal to Nur77antibody at a dilution of1:100(Abcam, Cambridge, MA, USA). Images were acquired and quantitated on an Olympus BX50microscope using Optimis software (Version6.2) and digitized using a color video camera (three-charge coupled device; JVC, Wayne, NJ). 4. Effect of Nur77on gene expression involved in intestinal lipid absorption in Caco-2cells by treatment with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmid over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77) and in apoE-/-mice by treatment with Nur77agonist, lentivirus encoding mouse Nur77(Ad-Nur77) and lentivirus encoding mouse Nur77(si-Nur77).4.1Caco-2cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing10%FCS with streptomycin (100μg/mL) and penicillin (100U/ml). All cells were incubated at37℃,5%CO2. Cells were seeded in6-or12-well plates or60-mm dishes and grown to80-90%confluence before use.Caco2cells by treatment with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmid over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77).4.2Total RNA from cultured cells was extracted using TRIzol reagent (Invitrogen) in accordance with the manufacturer’s instructions. Real-time quantitative PCR, using SYBR Green detection chemistry, was performed on the ABI7500Fast Real Time PCR system (Applied Biosystems, Foster City, CA, USA). Melt curve analyses of all real-time PCR products were performed and shown to produce a single DNA duplex. All samples were measured in triplicate and the mean value was considered for comparative analysis. Quantitative measurements were determined using the AACt method and GAPDH expression was used as the internal control.5. Effect of Nur77on plaque formation in ApoE-/-Mice by treatment with Nur77agonist, lentivirus encoding mouse Nur77(Ad-Nur77) and lentivirus encoding mouse Nur77(si-Nur77).5.1For en face analysis, aortas from different groups were opened longitudinally from the heart to the iliac arteries, and lesions were stained with Oil Red O. En face aortic lesion areas were digitized by a Nikon S6digital camera, analyzed using Image-Pro Plus image analysis software (Media Cybernetics, Bethesda, MD, USA), and expressed as the percentage of the total aortic surface area covered by lesions.5.2The upper portion of the heart and proximal aorta were obtained, embedded in Optimal Cutting Temperature (OCT) compound (Fisher, Tustin, CA), and stored at -70℃. Serial10-μm thick cryosections of aorta, beginning at the aortic root, were collected over a distance of400μm. Sections were stained with Oil Red O. The Oil Red O-positive areas in digitized color images of stained aortic root sections (three equally spaced sections per mouse; n=5per group) were quantified using Image-Pro Plus image analysis software (Media Cybernetics), and the data are expressed as percent of total section area.Results1. Nur77contributes to lipid loading, lipid content and cholesterol effluxWe first investigated the role of Nur77during lipid loading in THP-1macrophages, the effects of Nur77on lipid content and cholesterol efflux in THP-1macrophage-derived foam cells by treatment with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmid over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77). DiI-labeled ox-LDL uptake was obviously decreased by Csn-B and Ad-Nur77(P<0.001), while obviously increased by si-Nur77(P<0.001). Next, we examined the effects of Nur77on cholesterol content and cholesterol efflux in THP-1macrophage-derived foam cells by HPLC and liquid scintillation counting assays, respectively. Cellular cholesterol content was decreased while cholesterol efflux was increased when cells were treated with Csn-B and Ad-Nur77(P<0.05). On the contrary, cellular cholesterol content was increased while cholesterol efflux was decreased when cells were treated with si-Nur77(P<0.05).Subsequently, we performed mRNA expression analysis of a panel of genes involved in lipid uptake, lipid transport and cholesterol efflux in THP-1macrophage-derived foam cells by treatment with Csn-B, recombinant plasmid over-expressing Nur77and siRNAs against Nur77. Nur77up-regulated genes included ATP-binding cassette A1(ABCA1), scavenger receptor class B type1(SR-B1), Niemann-Pick C1protein (NPC1), Caveolin-1(CAV-1), neutral cholesterol ester hydrolase (nCEH)(P<0.05). Down-regulated genes through the expression of Nur77included low density lipoprotein receptor (LDLR), cholesteryl ester transfer protein (CETP), scavenger receptor CD36and SRA1(P<0.05). In addition, Nur77 had no effect on ATP-binding cassette G1(ABCG1) mRNA expression (P>0.05).Next, we explored the effect of Nur77on inflammatory gene expression in THP-1macrophage-derived foam cells by treatment with Csn-B, Ad-Nur77and si-Nur77. Nur77up-regulated genes included transforming growth factor (TGF-(3) and CD40(P<0.05), whereas down-regulated genes through the expression of Nur77included interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-12(IL-12), tumor necrosis factor-a (TNF-a), C-reactive protein (CRP), intercellular adhesion molecule-1(ICAM-1) and nuclear factor κB (NF-κB)(P<0.05). In addition, Csn-B had no effect on vascular cell adhesion molecule (VCAM-1) mRNA expression (P>0.05), but up-regulated interleukin-18(IL-18) mRNA expression (P<0.05). However, VCAM-1mRNA expression was inhibited by Ad-Nur77(P<0.05), while enhanced by Si-Nur77(P<0.05). Treatment with both Ad-Nur77and Si-Nur77had no effect on IL-18mRNA expression (P>0.05). Furthermore, the gene expressions of interferon-y (INF-y) and interleukin-10(IL-10) were not detectable in THP-1macrophage-derived foam cells through treatment with Csn-B, Ad-Nur77and si-Nur77.2. Nur77regulates plasma lipid parameters and circulating cytokine levels in apoE-/-MiceWe examined the terminal plasma lipid levels from experimental mice.the plasma high density lipoprotein cholesterol (HDL-C) showed a moderate reduction in the Csn-B group and Ad-Nur77group as compared to their control groups, respectively (P<0.05). Concomitantly, plasma low density lipoprotein cholesterol (LDL-C) increased in the Csn-B group and Ad-Nur77group as compared to their control groups, respectively (P<0.05). In contrast, treatment with si-Nur77led to an increase in plasma HDL-cholesterol (P<0.05) and a reduction in plasma LDL-cholesterol as compared to the si-Mock group (P<0.05). However, no significant alternation occurred in apoAl, apolipoprotein B (apoB), very low density lipoprotein cholesterol (VLDL-cholesterol), total triglyceride and cholesterol (P>0.05).To investigate whether change in Nur77expression could result in corresponding changes in plasma inflammatory cytokines, we conducted a series of ELISAs. Consistent with the data of inflammatory gene expression in THP-1macrophage-derived foam cells, treatment with both Csn-B and Ad-Nur77resulted in down-regulation of CRP concentrations in plasma, respectively (P<0.05). In agreement with these data, si-Nur77-mediated knockdown of Nur77results in an up-regulation of CRP concentrations in plasma (P<0.05). However, no significant alteration occurred in levels of IL-1β, IL-6and TNF-a between groups at the end of the experiments (P>0.05).3. Nur77affects hepatic lipid deposition in apoE-/-miceThe protein expression of Nur77in mouse liver was investigated by Western blot and immunohistochemistry analyses. We observed that minor expression levels were detectable in the baseline group, but expression of Nur77was markedly increased in the control group. In addition, the Csn-B group and Ad-Nur77group had significantly higher expression of Nur77than their control groups while the si-Nur77group had a lower expression of Nur77as compared to the si-Mock group. Next, the effects of Nur77on lipid content in the liver of apoE-/-mice were analyzed by Oil Red O staining and measured enzymatically. Hepatic triglyceride levels in the liver was reduced in mice treated with Csn-B and Ad-Nur77, respectively, compared with mice of the control group (P<0.05). In contrast, the hepatic triglyceride levels in the liver was increased in mice treated with si-Nur77in comparison to mice treated with si-Mock (P<0.05). However, hepatic cholesterol level had no change in response to regulation of expression of Nur77in the liver (P>0.05).To investigate the mechanisms of Nur77reduction of hepatic lipid deposition, hepatic lipid metabolism gene expression levels in livers of apoE-/-mice were analyzed by real time PCR. As shown, Csn-B and Ad-Nur77treatment progressively reduced gene levels of LDLR, ATP-binding cassette G5(ABCG5), CRP, SREBP1C, SREBP2(P<0.05), and increased gene levels of SR-B1and hepatic lipase (HL)(P<0.05). In addition, treatment with si-Nur77could up-regulated gene expression of LDLR, ABCG5, CRP, SREBP1C and SREBP2(P<0.05). While SR-B1and HL gene expressions were down-regulated by treatment with si-Nur77(P<0.05). Although gene expression of HMG-CoA reductase (HMGCR) was repressed by treatment with Csn-B (P<0.05), the gene expression of HMGCR was increased by treatment with Ad-Nur77(P<0.05) while decreased by treatment with si-Nur77(P<0.05). In addition, Nur77had no effect on apoA1and LCAT expression (P>0.05).4. Nur77inhibits gene expression involved in intestinal lipid absorptionTo address the possibility that expression levels of intestinal lipid transporters are affected by Nur77, we incubated Caco-2cells with Csn-B, recombinant plasmid Ad-Nur77and siRNAs against Nur77(si-Nur77). Csn-B and Ad-Nur77treatment progressively reduced gene levels of microsomal triglyceride transfer protein (MTP), Niemann-Pick Cl-like1(NPC1L1) and ABCG5(P<0.05). On the contrary, si-Nur77treatment markedly induced gene expression of MTP, NPC1L1and ABCG5(P<0.05). To further investigate the Nur77mechanisms affecting intestinal cholesterol absorption in apoE-/-mice, gene expression in intestinal tissue of apoE-/-mice were analyzed by real time PCR. We also found that over-expression of Nur77reduced MTP, NPC1L1and ABCG5gene expression (P<0.05), while silenced Nur77increased these gene levels (P<0.05).5. Nur77reduces plaque formation in ApoE-/-MiceTo investigate the impact of Nur77on atherogenesis in apoE-/-mice, atherosclerotic lesions were evaluated by aortic valve section and en face analyses. Mice receiving Csn-B and Ad-Nur77showed a decrease in the average lesion area compared with their controls by both en face and aortic valve section analyses (P<0.05). On the contrary, si-Nur77-treated mice showed an increase in average lesion area compared with si-Mock-treated mice by both en face and aortic valve section analyses (P>0.05).Next, the gene expression changes of the inflammatory molecules, adhesion molecules and molecules related to cholesterol metabolism were investigated in aortic tissues. In both Csn-B-treated and Ad-Nur77-treated apoE-/-mice, gene expression of SRA1, CD36, IL-1β, IL-6, CRP, ICAM-1, macrophage inflammatory protein-1α (MIP-1α) and monocyte chemo-attractant protein-1(MCP-1) were markedly repressed (P<0.05), but gene expression of ABCA1, SR-B1, NPC1and CD40were up-regulated at week12(P<0.05). On the contrary, we found that gene expression of SRA1, CD36, IL-1β, IL-6, CRP, ICAM-1, MIP-1α and MCP-1were up-regulated (P<0.05), while gene expression of ABCA1, SRB1, NPC1and CD40were down-regulated (P<0.05) in the aorta of si-Nur77-treated ApoE-/-mice. In addition, gene expression of TNF-a was reduced in Ad-Nur77-treated ApoE-/-mice (P<0.05), while increased in si-Nur77-treated ApoE-/-mice (P<0.05). However, Csn-B-treated apoE-deficient mice had no change in TNF-a gene expression (P>0.05).Conclusions1. Nur77reduces lipid loading, lipid content and enhanced cholesterol efflux in THP-1macrophage-derived foam cells.2. Nur77decreases plasma HDL-cholesterol level, increases plasma LDL-cholesterol level and decreases CRP concentrations in apoE-/-Mice.3. Nur77down-regulates triglyceride levels but not cholesterol level in the liver.4. Nur77inhibits gene expression involved in intestinal lipid absorption.5. Nur77reduces plaque formation in ApoE-/-Mice. |
PDF Full Text Request