Hypercholesterolaemia and inflammation are correlated with atherogenesis. Orphan nuclear receptor NR4A1, as a key regulator of inflammation, is closely associated with lipid levels in vivo. However, the mechanism by which lipids regulate NR4A1 expression remains unknown. We aimed to elucidate the underlying mechanism of NR4A1 expression in monocytes during hypercholesterolaemia, and reveal the potential role of NR4A1 in hypercholesterolaemia-induced circulating inflammation. Circulating leucocytes were collected from blood samples of 139 patients with hypercholesterolaemia and 139 sex- and age-matched healthy subjects. We found that there was a low-grade inflammatory state and higher expression of NR4A1 in patients. Both total cholesterol and low-density lipoprotein cholesterol levels in plasma were positively correlated with NR4A1 mRNA level. ChIP revealed that acetylation of histone H3 was enriched in the NR4A1 promoter region in patients. Human mononuclear cell lines THP-1 and U937 were treated with cholesterol. Supporting our clinical observations, cholesterol enhanced p300 acetyltransferase and decreased HDAC7 (histone deacetylase 7) recruitment to the NR4A1 promoter region, resulting in histone H3 hyperacetylation and further contributing to NR4A1 up-regulation in monocytes. Moreover, cytosporone B, an NR4A1 agonist, completely reversed cholesterol-induced IL-6 (interleukin 6) and MCP-1 (monocyte chemoattractant protein 1) expression to below basal levels, and knockdown of NR4A1 expression by siRNA not only mimicked, but also exaggerated the effects of cholesterol on inflammatory biomarker up-regulation. Thus we conclude that histone acetylation contributes to the regulation of NR4A1 expression in hypercholesterolaemia, and that NR4A1 expression reduces hypercholesterolaemia-induced inflammation.

CLINICAL PERSPECTIVES

  • Strong evidence suggests a synergy between inflammation and hypercholesterolaemia in atherosclerosis. NR4A1, a key regulator of inflammation, is closely associated with lipid levels in vivo. However, the molecular regulation of NR4A1 expression by hypercholesterolaemia and the potential role of NR4A1 in hypercholesterolaemia-induced inflammation are largely unknown.

  • Results of the present study show that NR4A1 is significantly induced by hypercholesterolaemia through enhancing histone acetylation in the promoter region of NR4A1. NR4A1 mediates self-protection responses in hypercholesterolaemia-induced inflammation in monocytes.

  • These findings provide new evidence that NR4A1 reduces hypercholesterolaemia-induced inflammation and may represent a potential molecular target for the prevention and treatment of hypercholesterolaemia-associated atherosclerosis.

INTRODUCTION

Atherosclerosis is a chronic inflammatory disease, which is a major cause of death worldwide. Hypercholesterolaemia, as one of the major risks of atherosclerosis, results in higher circulating concentrations of inflammatory biomarkers, such as IL (interleukin)-6 and MCP-1 (monocyte chemoattractant protein 1) [1]. Conversely, chronic inflammation has been increasingly recognized as a major contributor to the development and progression of atherosclerosis [2]. Evidence from clinical trials indicates that hypercholesterolaemia therapy is associated with decreasing concentrations of inflammatory mediators, such as CRP (C-reactive protein), IL-6 or MCP-1, and leads to reductions in the incidence of death from cardiovascular disease [36]. To date, several NRs (nuclear receptors), such as peroxisome-proliferator-activated receptors [7], liver X receptors [8] and NR4As [9], have been proposed as emerging players in the modulation of inflammation and atherosclerosis. At the molecular level, previous research has identified effects of hypercholesterolaemia on chromatin epigenetic modification [1013]. In this regard, modification of chromatin is considered to be a mechanism for long-term hypercholesterolaemia-induced cardiovascular diseases.

NR4A1 (also referred to as NGFI-B, Nur77 or TR3), a member of the NR4A orphan nuclear receptor superfamily, is known to be an immediate-early gene that responds to stimuli such as peptide hormones, lipopolysaccharide, oxidized lipoproteins and various forms of physical stimulation [14]. Most studies on the mechanism by which NR4A1 is regulated have focused principally on post-translational modifications, such as phosphorylation [15,16], acetylation and ubiquitylation [17]. Despite studies reporting that NR4A1 is regulated by histone acetylation [18,19], little is known about the transcriptional regulation of NR4A1. Accumulating data demonstrate that NR4A1 is expressed in multiple cell types of crucial importance in immunity and in chronic inflammation of the vessel wall involved in atherosclerosis [14]. Of clinical relevance, NR4A1 is localized within macrophages in human atherosclerotic lesions, reduces macrophage lipid loading and inhibits the production of pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and MCP-1) [20]. NR4A1 deficiency causes macrophage polarization into a pro-inflammatory phenotype and increases atherosclerosis [2123]. This pro-inflammatory response, due to NR4A1 deletion, results from increasing Toll-like receptor expression and enhanced NF-κB (nuclear factor κB) activity in monocytes and macrophages [21,24]. In addition, NR4A1 is highly expressed in Ly6C monocytes, and functions as a master regulator of the differentiation and survival of the Ly6C monocyte subset [24]; the latter patrol the resting vasculature and participate in the resolution of inflammation [25]. These findings suggest that NR4A1 may have a protective role in inflammation.

Previous studies indicate that NR4A1 expression correlates with changes in fat mass in both rodents and humans [26,27]. NR4A1 expression is 23-fold higher in adipose tissue from obese patients and becomes normal after fat loss [27]. These results suggest an effect of fat mass on NR4A1 expression. However, the molecular mechanism by which NR4A1 expression is regulated by lipids, especially the relationship between NR4A1 expression and hypercholesterolaemia, remains largely unknown. Moreover, the potential role for NR4A1 in hypercholesterolaemia-induced inflammation and the underlying mechanism, also needs further study, and will provide a novel theoretical basis and potential therapeutic target for hypercholesterolaemia-associated atherosclerosis. Therefore the aim of the present study was to investigate the regulation of NR4A1 expression in hypercholesterolaemia, and identify the potential role of NR4A1 in hypercholesterolaemia-induced circulating inflammation. We hypothesized that hypercholesterolaemia induces epigenetic alterations that modulate NR4A1 expression in monocytes, and that NR4A1 functions as an inhibitor that limits the intensity and duration of hypercholesterolaemia-induced inflammation in hypercholesterolaemia patients.

MATERIALS AND METHODS

Participants

We performed a case-control study of 139 hypercholesterolaemia patients who were visiting the First Affiliated Hospital of Shantou University Medical College for a health examination. Patients were characterized according to the American Association of Clinical Endocrinologists’ Guidelines for Management of Dyslipidaemia and Prevention of Atherosclerosis: total cholesterol ≥240 mg/dl (6.22 mmol/l), which is considered to be the high-risk serum lipid concentration [28]. In all subjects, background information was collected, including lifestyle and medical history. None of the patients had a history of hypertension, coronary heart disease, diabetes mellitus, stroke, hepatic or kidney disease, cancer or treatment with anti-inflammatory drugs. We used SPSS 17.0 to randomly select 139 sex- and age-matched healthy subjects as the healthy control group.

Approximately 1.5 ml of venous blood was drawn from all subjects into EDTA vacuum tubes after ≥8 h of fasting. After centrifugation at 500 g for 15 min, plasma was separated into a new tube for biochemical measurements. Leucocyte cell samples were separated into new tubes for DNA, RNA and protein analysis respectively. Plasma and leucocytes were stored at −80°C until use.

All patients provided informed written consent in this study, and the study protocol was approved by the local ethics committees (Shantou University Medical College and the First Affiliated Hospital of Shantou University Medical College, China). All procedures were performed according to the Declaration of Helsinki and Good Clinical Practice guidelines.

Cell culture

Human mononuclear cell lines THP-1 and U937 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cell lines were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS (Hyclone) in a humidified 37°C incubator with 5% CO2. Cholesterol (20 mg/ml in 100% ethanol) (Sigma) [2931] was prepared fresh before incubation and was added directly to the medium to a final concentration of 40 or 60 μg/ml. CsnB (cytosporone B) (Santa Cruz Biotechnology) in DMSO was used at a final concentration of 10 μg/ml.

Biochemical measurements

Biochemical variables, including cholesterol, HDL-C (high-density lipoprotein cholesterol), LDL-C (low-density lipoprotein cholesterol), TRIG (triacylglycerol), fasting blood glucose and high-sensitivity CRP were measured by an enzymatic method using an LX-20 automatic biochemistry analyser (Beckman Coulter). WBC (white blood cell) and RBC (red blood cell) counts in blood were determined with a Coulter ACT.5Diff Hematology Analyzer (Beckman Coulter) in the clinical laboratory of the First Affiliated Hospital of Shantou University Medical College. All reagents were from Olympus Diagnostics.

qRT-PCR (quantitative real-time reverse transcription–PCR)

Total RNA was extracted using TRIzol® (Invitrogen) and was reverse-transcribed with a QuantiTect Reverse Transcription kit (Qiagen) for cDNA synthesis and genomic DNA removal. qRT-PCR was performed using a SYBR Green PCR mix kit (TaKaRa Bio) and carried out using an Applied Biosystems Prism 7500 instrument. Expression of analysed genes was normalized to β-actin. All qRT-PCR primers used are listed in Supplementary Table S1.

Western blot analysis

Leucocyte cell samples and treated human mononuclear cells were lysed in RIPA buffer supplemented with 1% protease inhibitor cocktail (Millipore). Protein concentration was determined with a BCA protein assay kit (NovasyGen). Equal amounts of protein (30 μg) underwent SDS/PAGE (10% gel) and were transferred on to a nitrocellulose membrane (Millipore). Immunoblotting involved primary antibodies against NR4A1 (Santa Cruz Biotechnology) and β-actin (Sigma). Corresponding secondary antibodies were applied and blots were developed by use of a Super ECL Plus Detection Reagent (NovasyGen). The intensity of bands was determined by ImageJ 2X software (NIH) and normalized to β-actin.

Cholesterol measurement

After being treated with cholesterol for 1 h, THP-1 cells were washed three times in ice-cold PBS, then whole-cell lysates were collected for total cholesterol measurement by enzymatic assay according to the manufacturer's instructions (Total Cholesterol Assay Kit, Nanjing Jiancheng Bioengineering Institute). Protein concentration was determined with a BCA protein assay kit. The cholesterol concentration was normalized to protein concentration [30].

ELISA

Protein levels of MCP-1 in plasma and cell culture supernatant were separately detected by use of a Quantikine® Human MCP-1 Immunoassay (ELISA) Kit (R&D Systems) and Human MCP-1 ELISA kit (Neobioscience Technology). The level of IL-6 was determined by a RayBio® Human IL-6 ELISA kit (RayBiotech) and Valukine™ Human IL-6 Immunoassay (ELISA) Kit (R&D Systems) respectively.

ChIP assay

As we described previously [32], ChIP was performed with EZ-Magna ChIP A and EZ-Magna ChIP G Kits (Millipore) according to the manufacturer's protocol. ChIP-grade antibodies were as follows: anti-acetyl-histone H3, anti-RNA polymerase II, anti-p300, anti-HDAC7 (histone deacetylase 7) and normal rabbit IgG (Abcam). Immunoprecipitated DNA was analysed by qRT-PCR normalized with the input DNA. Primer sequences in reference to the promoter region of NR4A1 are listed in Supplementary Table S1.

RNAi assay

RNAi was used to knockdown the expression of NR4A1 in THP-1 cells. siRNA for NR4A1 and a scrambled RNA were obtained from GenePharma. THP-1 cells were transfected with 100 pmol of siRNA by 5 μl of Lipofectamine™ 2000 reagent (Invitrogen) for 48 h in six-well plates. To confirm the effect of siRNA on the expression of NR4A1, transfected cells underwent qRT-PCR and Western blot analysis. siRNA primer sequences are specified in Supplementary Table S1.

Statistical analysis

Analyses were performed using SPSS 17.0. An independent-sample Student's t test (unpaired) was used to compare continuous variables and a χ2 test was used for categorical variables. NR4A1 mRNA levels from subjects were log-transformed to normalize their skewed distributions and are denoted as Lg-NR4A1. Partial correlation and linear regression analysis were used to evaluate the association of lipid levels and Lg-NR4A1 mRNA expression after adjusting for age (in years) and sex (male or female). Cell experiments were performed in at least three independent experiments. Results in the text and the Tables are given as means±S.D. and in the Figures as means±S.E.M. All statistical significance was defined as P<0.05 for two-tailed analysis.

RESULTS

Population characteristics of the study participants

A summary of the clinical parameters and laboratory values of the participants is shown in Table 1. We included 278 participants consisting of 139 patients and 139 age- and sex-matched healthy controls. Compared with controls, hypercholesterolaemia patients had higher plasma levels of cholesterol, LDL-C and TRIG. As expected, for patients, a statistically significant increase was also observed in inflammatory biomarkers: high-sensitivity CRP, MCP-1 and IL-6, in both plasma and peripheral blood leucocytes (Table 1 and Supplementary Figure S1). This suggested that there was both a disorder of lipid metabolism and a chronic low-grade inflammatory state in patients with hypercholesterolaemia. BMI (body mass index), blood pressure, HDL-C, glucose, WBC counts and RBC counts were similar between the two groups.

Table 1
Population characteristics of hypercholesterolaemia patients and controls

Data are given as means (±S.D.) unless indicated as n (%). Bold italic indicates significant difference.

Variable§Control (n=139)Hypercholesterolaemia (n=139)P value
Age (year) 51.91 (11.58) 53.17 (11.30) 0.360 
Male (n,%)  65 (46.8%)  56 (40.3%) 0.333 
BMI (kg/m223.05 (3.07) 23.84 (4.19) 0.086 
Blood pressure (mmHg)    
 SBP 118.98 (11.70) 119.31 (11.43) 0.818 
 DBP 76.47 (8.89) 77.90 (8.65) 0.180 
Cholesterol (mmol/l) 4.61 (0.69) 7.42 (0.80) 0.000 
LDL-C (mmol/l) 2.71 (0.63) 5.17 (0.75) 0.000 
HDL-C (mmol/l) 1.40 (0.35) 1.45 (0.39) 0.222 
TRIG (mmol/l) 1.03 (0.50) 2.42 (2.51) 0.000 
Glucose (mmol/l) 5.28 (0.38) 5.19 (0.40) 0.066 
WBC (×109/l) 6.74 (1.96) 7.03 (1.65) 0.187 
RBC (×1012/l) 4.68 (0.47) 4.71 (0.53) 0.639 
hsCRP (mg/l) 1.34 (2.38) 2.44 (3.93) 0.037 
MCP-1 (pg/ml) 146.88 (51.68) 167.81 (53.90) 0.016 
IL-6 (pg/ml) 1.39 (1.23) 2.96 (4.27) 0.021 
Variable§Control (n=139)Hypercholesterolaemia (n=139)P value
Age (year) 51.91 (11.58) 53.17 (11.30) 0.360 
Male (n,%)  65 (46.8%)  56 (40.3%) 0.333 
BMI (kg/m223.05 (3.07) 23.84 (4.19) 0.086 
Blood pressure (mmHg)    
 SBP 118.98 (11.70) 119.31 (11.43) 0.818 
 DBP 76.47 (8.89) 77.90 (8.65) 0.180 
Cholesterol (mmol/l) 4.61 (0.69) 7.42 (0.80) 0.000 
LDL-C (mmol/l) 2.71 (0.63) 5.17 (0.75) 0.000 
HDL-C (mmol/l) 1.40 (0.35) 1.45 (0.39) 0.222 
TRIG (mmol/l) 1.03 (0.50) 2.42 (2.51) 0.000 
Glucose (mmol/l) 5.28 (0.38) 5.19 (0.40) 0.066 
WBC (×109/l) 6.74 (1.96) 7.03 (1.65) 0.187 
RBC (×1012/l) 4.68 (0.47) 4.71 (0.53) 0.639 
hsCRP (mg/l) 1.34 (2.38) 2.44 (3.93) 0.037 
MCP-1 (pg/ml) 146.88 (51.68) 167.81 (53.90) 0.016 
IL-6 (pg/ml) 1.39 (1.23) 2.96 (4.27) 0.021 

NR4A1 expression is increased in leucocytes from hypercholesterolaemia patients

We then tested whether NR4A1 expression was significantly different between the two groups. As shown in Figure 1, NR4A1 mRNA expression levels were elevated more than 3-fold in peripheral blood leucocytes from hypercholesterolaemia patients, compared with healthy controls (125.34±25.91 compared with 37.77±8.86, P<0.01; Figure 1A). Consistent with elevated NR4A1 mRNA, protein levels of NR4A1 in patients were also increased (7.93±6.76 compared with 4.38±3.71, P<0.01; Figure 1B). Thus plasma levels of lipid may be correlated with NR4A1 expression.

NR4A1 mRNA and protein expression in peripheral blood leucocytes from controls and patients

Figure 1
NR4A1 mRNA and protein expression in peripheral blood leucocytes from controls and patients

(A) qRT-PCR analysis of mRNA expression with β-actin as the internal control. (B) Western blot analysis of protein expression with β-actin as the internal control. Quantification of protein levels was by densitometry. Results are means±S.E.M. **P<0.01 compared with controls.

Figure 1
NR4A1 mRNA and protein expression in peripheral blood leucocytes from controls and patients

(A) qRT-PCR analysis of mRNA expression with β-actin as the internal control. (B) Western blot analysis of protein expression with β-actin as the internal control. Quantification of protein levels was by densitometry. Results are means±S.E.M. **P<0.01 compared with controls.

Next, we investigated the relationship between NR4A1 expression and blood lipid profile (cholesterol, LDL-C, HDL-C and TRIG). Given that age has an important influence on NR4A1 expression [26], we performed analysis using a partial correlation and found the Lg-NR4A1 mRNA level to be positively correlated with age (r=0.29, P<0.001; Supplementary Figure S2). In addition, Lg-NR4A1 mRNA was significantly and positively associated with plasma levels of cholesterol (r=0.31, P<0.001; Figure 2A) and LDL-C (r=0.30, P<0.001; Figure 2B), but no characteristics were associated with HDL-C (P=0.078; Figure 2C) or TRIG (P=0.103; Figure 2D), after adjustment for sex, age, BMI, blood pressure, WBC, RBC and glucose.

Correlation of log-transformed NR4A1 mRNA levels and cholesterol (A), LDL-C (B), HDL-C (C) and TRIG (D) levels

Figure 2
Correlation of log-transformed NR4A1 mRNA levels and cholesterol (A), LDL-C (B), HDL-C (C) and TRIG (D) levels

Results were adjusted for sex, age, BMI, blood pressure, WBC counts, RBC counts and glucose in controls and patients. Controls are shown as blue squares (n=71) and patients as red circles (n=81).

Figure 2
Correlation of log-transformed NR4A1 mRNA levels and cholesterol (A), LDL-C (B), HDL-C (C) and TRIG (D) levels

Results were adjusted for sex, age, BMI, blood pressure, WBC counts, RBC counts and glucose in controls and patients. Controls are shown as blue squares (n=71) and patients as red circles (n=81).

We studied further the effect of blood lipid profile on the Lg-NR4A1 mRNA level by linear regression analysis. Table 2 shows that the levels of cholesterol (P<0.001), LDL-C (P<0.001) and TRIG (P=0.045), but not HDL-C (P=0.086), correlate with Lg-NR4A1 mRNA levels following adjustment for age and sex. Therefore elevated levels of cholesterol and LDL-C contribute to the increase in NR4A1 expression in leucocytes from hypercholesterolaemia patients.

Table 2
Linear regression analysis of association between lipid levels and Lg-NR4A1 mRNA expression adjusted for age and sex

Bold italic indicates significant difference. CI, confidence interval.

Lg-NR4A1 mRNA level
Lipid profileB95% CIP value
Cholesterol (mmol/l) 0.161 (0.085, 0.236) 0.000 
LDL-C (mmol/l) 0.181 (0.092, 0.269) 0.000 
HDL-C (mmol/l) 0.377 (−0.054, 0.809) 0.086 
TRIG (mmol/l) 0.201 (0.004, 0.397) 0.045 
Lg-NR4A1 mRNA level
Lipid profileB95% CIP value
Cholesterol (mmol/l) 0.161 (0.085, 0.236) 0.000 
LDL-C (mmol/l) 0.181 (0.092, 0.269) 0.000 
HDL-C (mmol/l) 0.377 (−0.054, 0.809) 0.086 
TRIG (mmol/l) 0.201 (0.004, 0.397) 0.045 

Acetylated histone H3 is enriched in the promoter region of the NR4A1 gene in hypercholesterolaemia patients

Hypercholesterolaemia can affect epigenetic patterning in the vasculature [10], and HDAC inhibition has been shown to strongly up-regulate transcription of NR4A1 [18]. This suggests that hypercholesterolaemia may induce epigenetic alterations that modulate NR4A1 expression in peripheral blood leucocytes. Subsequently, we determined the acetylation status of histone H3 (Ace-H3) in the promoter region of the NR4A1 gene using ChIP. Compared with controls, Ace-H3 was enriched in the NR4A1 promoter region of leucocytes from hypercholesterolaemia patients (5.00±4.05 compared with 2.79±1.73, P<0.05; Figure 3). Given that Ace-H3 has been reported to be associated with active genes [33], we predict that hypercholesterolaemia may induce epigenetic activation, thereby increasing NR4A1 mRNA expression in leucocytes.

Histone H3 acetylation patterns in the NR4A1 promoter region in controls and patients

Figure 3
Histone H3 acetylation patterns in the NR4A1 promoter region in controls and patients

(A) Schematic structure of the design for the ChIP assay of the NR4A1 promoter. TSS, transcription start site. (B) ChIP analysis of Ace-H3 enrichment in the NR4A1 gene promoter. ChIP enrichment was measured using qRT-PCR, normalized by the input DNA. Results are means±S.E.M. *P<0.05 compared with controls.

Figure 3
Histone H3 acetylation patterns in the NR4A1 promoter region in controls and patients

(A) Schematic structure of the design for the ChIP assay of the NR4A1 promoter. TSS, transcription start site. (B) ChIP analysis of Ace-H3 enrichment in the NR4A1 gene promoter. ChIP enrichment was measured using qRT-PCR, normalized by the input DNA. Results are means±S.E.M. *P<0.05 compared with controls.

Histone acetylation is involved in cholesterol-induced NR4A1 expression in vitro

To elucidate further the molecular mechanism of hypercholesterolaemia-associated NR4A1 up-regulation, we treated human THP-1 mononuclear cell with cholesterol (40 μg/ml). Under these conditions, cells had normal morphology and no obvious apoptosis was detected (Supplementary Figures S3A and S3B). Cholesterol loading increased the total cellular cholesterol level by approximately 80% (Figure 4A). qRT-PCR and Western blot analysis showed that cholesterol treatment up-regulated NR4A1 expression (Figures 4B and 4C). When cells were treated with cholesterol, the mRNA and protein levels of NR4A1 increased 2.6-fold and 2.0-fold respectively. Consistent with an increase in NR4A1 transcription, cholesterol increased the recruitment of RNA pol (polymerase) II in the NR4A1 promoter (Figure 4D).

Cholesterol-induced histone H3 acetylation in the NR4A1 promoter contributes to NR4A1 up-regulation in vitro

Figure 4
Cholesterol-induced histone H3 acetylation in the NR4A1 promoter contributes to NR4A1 up-regulation in vitro

THP-1 cells were treated with ethanol (Ctrl) or cholesterol (CHL) (40 μg/ml) for 1 h. (A) Cells were lysed and cholesterol levels were determined from total cell lysates. (B) qRT-PCR analysis of mRNA expression for NR4A1 with β-actin as the internal control. (C) Western blot analysis of protein expression with β-actin as the internal control. Quantification of protein levels was by densitometry. (DG) ChIP analysis of RNA pol II (D), Ace-H3 (E), p300 (F) and HDAC7 (G) enrichment in the NR4A1 gene promoter. ChIP enrichment was measured using qRT-PCR, normalized by the input DNA. Results are means±S.E.M. for three independent experiments. **P<0.01 compared with control, *P<0.05 compared with control.

Figure 4
Cholesterol-induced histone H3 acetylation in the NR4A1 promoter contributes to NR4A1 up-regulation in vitro

THP-1 cells were treated with ethanol (Ctrl) or cholesterol (CHL) (40 μg/ml) for 1 h. (A) Cells were lysed and cholesterol levels were determined from total cell lysates. (B) qRT-PCR analysis of mRNA expression for NR4A1 with β-actin as the internal control. (C) Western blot analysis of protein expression with β-actin as the internal control. Quantification of protein levels was by densitometry. (DG) ChIP analysis of RNA pol II (D), Ace-H3 (E), p300 (F) and HDAC7 (G) enrichment in the NR4A1 gene promoter. ChIP enrichment was measured using qRT-PCR, normalized by the input DNA. Results are means±S.E.M. for three independent experiments. **P<0.01 compared with control, *P<0.05 compared with control.

Since acetylation of histone H3 by HAT (histone acetyltransferase) p300 is thought to be a unique hallmark of gene activation and deacetylation of histone H3 by HDAC is an epigenetic modification generally associated with gene silencing, we examined the effect of cholesterol treatment on the acetylation status of histone H3 in the promoter region of NR4A1. Cholesterol nearly doubled the level of Ace-H3 in the NR4A1 promoter, consistent with our clinical trial results (Figure 4E). Consistently, we observed an approximate 2.0-fold increase in p300 and 38.3% decrease in HDAC7 recruitment in the NR4A1 promoter region after cholesterol treatment (Figures 4F and 4G). Similarly, cholesterol-induced NR4A1 up-regulation was confirmed in the human U937 mononuclear cell line (Supplementary Figure S4). Thus the clinical and in vitro data indicate that cholesterol affected epigenetic modification, at least in part, through inducing histone H3 hyperacetylation, thereby contributing to NR4A1 transcriptional activation in monocytes.

NR4A1 prevents cholesterol-induced inflammation

Previous studies have indicated that NR4A1 exerts effects on inflammatory processes [20,34]. To investigate the potential role of NR4A1 in hypercholesterolaemia-induced inflammation, we explored the effect of NR4A1 on inflammatory gene expression in THP-1 cells following treatment with cholesterol (60 μg/ml), before or after treatment with NR4A1 siRNA or an NR4A1 agonist CsnB (10 μg/ml). CsnB, as a specific NR4A1 agonist, stimulates the expression and specifically promotes transactivation of NR4A1 [35]; it is a useful tool to increase understanding of NR4A1 biological function. Consistent with previous studies, CsnB at a concentration of 10 μg/ml increased the mRNA and protein expression level of NR4A1, and did not prompt THP-1 apoptosis in the present study (Supplementary Figures S5A–S5D). We found that the mRNA expression of both IL-6 and MCP-1 were increased by cholesterol, whereas expression of both was attenuated by CsnB (Figures 5A and 5B). Furthermore, CsnB completely reversed cholesterol-induced IL-6 and MCP-1 expression to below basal levels. Intriguingly, knockdown of NR4A1 by siRNA for 48 h, which decreased NR4A1 expression to 50% (Supplementary Figures S5E and S5F), resulted in approximate 5.0- and 3.0-fold increases in mRNA levels of IL-6 and MCP-1 respectively, and correspondingly elevated cholesterol-induced IL-6 and MCP-1 expression (Figures 5C and 5D). The change in IL-6 and MCP-1 protein levels also strongly supports the above results (Figures 5E–5H). Thus our results indicate that cholesterol induces NR4A1 transcription in monocytes, which functions as an inhibitor to limit the intensity and duration of hypercholesterolaemia-induced inflammation in hypercholesterolaemia patients.

Effect of NR4A1 on cholesterol-induced IL-6 and MCP-1 expression

Figure 5
Effect of NR4A1 on cholesterol-induced IL-6 and MCP-1 expression

(A, B, E and F) THP-1 cells were treated with ethanol (Ctrl) or cholesterol (CHL) (60 μg/ml) or plus CsnB (10 μg/ml) for 24 h. (C, D, G and H) THP-1 cells were transfected with siRNA against NR4A1 (si-NR4A1) or non-targeting siRNA (si-mock) as control for 24 h, then were treated with cholesterol (60 μg/ml) for 24 h. qRT-PCR analysis of mRNA expression for IL-6 (A and C) and MCP-1 (B and D) with β-actin as the internal control. ELISA analysis of the concentration of IL-6 (E and G) and MCP-1 (F and H) in THP-1 cell supernatant. Results are means±S.E.M. for at least three independent experiments. **P<0.01 compared with control or si-mock, *P<0.05 compared with control or si-mock, ##P<0.01 compared with cholesterol, #P<0.05 compared with cholesterol.

Figure 5
Effect of NR4A1 on cholesterol-induced IL-6 and MCP-1 expression

(A, B, E and F) THP-1 cells were treated with ethanol (Ctrl) or cholesterol (CHL) (60 μg/ml) or plus CsnB (10 μg/ml) for 24 h. (C, D, G and H) THP-1 cells were transfected with siRNA against NR4A1 (si-NR4A1) or non-targeting siRNA (si-mock) as control for 24 h, then were treated with cholesterol (60 μg/ml) for 24 h. qRT-PCR analysis of mRNA expression for IL-6 (A and C) and MCP-1 (B and D) with β-actin as the internal control. ELISA analysis of the concentration of IL-6 (E and G) and MCP-1 (F and H) in THP-1 cell supernatant. Results are means±S.E.M. for at least three independent experiments. **P<0.01 compared with control or si-mock, *P<0.05 compared with control or si-mock, ##P<0.01 compared with cholesterol, #P<0.05 compared with cholesterol.

DISCUSSION

Strong evidence suggests a synergy between inflammation and hypercholesterolaemia in atherogenesis [1,3,36]. Prolonged hypercholesterolaemia exposure increases the expression of inflammatory mediators, and facilitates the development and progression of atherosclerosis. NR4A1, as a key regulator of inflammation, is closely associated with lipid level in vivo. Here, we define the role of NR4A1 in hypercholesterolaemia-induced inflammation and the underlying mechanism. Our novel findings are as follows: (i) high cholesterol affects epigenetic modification at the NR4A1 promoter, at least in part, through inducing histone H3 hyperacetylation, and up-regulates NR4A1 expression in monocytes in vivo and in vitro; (ii) increasing NR4A1 expression and activity suppresses cholesterol-induced MCP-1 and IL-6 expression; and (iii) NR4A1 suppression potentiates the stimulatory effects of cholesterol on MCP-1 and IL-6 expression and secretion.

The subjects who were diagnosed as having chronic disease or hypercholesterolaemia might intentionally change their lifestyle or be treated with drugs towards a healthier one. Moreover, the expression of NR4A1 and the inflammatory response are associated with diabetes in both mice and humans [37,38]. To avoid this type of bias, all patients included in our study were newly diagnosed with hypercholesterolaemia and had no other chronic diseases, including diabetes. The results of this study population support the evidence that hypercholesterolaemia is a common disorder characterized by chronic low-grade inflammation. The present study confirms that expression and secretion of inflammatory cytokines and chemokines (CRP, IL-6 and MCP-1) are significantly increased in peripheral blood leucocytes from patients, which is in agreement with previous clinical studies [1,3].

A few studies have suggested that the expression of NR4A1 is closely associated with lipids [8,26,27]. However, only one study investigated how lipids regulate NR4A1 expression [39], and focused on the effect of oxidized LDL on murine macrophages. Therefore no previous study, based on clinical data analysis, has yet assessed the association between hypercholesterolaemia and NR4A1 expression in leucocytes. In the present study, we demonstrate, for the first time, that NR4A1 expression is directly affected by hypercholesterolaemia in leucocytes. Plasma levels of both cholesterol and LDL-C strongly positively correlate with NR4A1 expression in peripheral blood leucocytes. Linear regression analysis further demonstrated that plasma levels of cholesterol and LDL-C significantly increase age- and sex-adjusted Lg-NR4A1 mRNA expression. The reasons behind the same effect of cholesterol and LDL-C on NR4A1 expression are clear primarily because LDL is the major plasma carrier of cholesterol in the form of cholesterol ester [40], and plasma levels of LDL-C are generally consistent with the concentration of cholesterol in human plasma.

Monocytes play a central role in atherosclerotic disease and inflammatory responses by producing cytokines [41]. Given the limitation of measuring the expression of NR4A1 in overall leucocyte populations, but not specific leucocyte subpopulations, we next examined the effect of high cholesterol on NR4A1 expression in human mononuclear cell lines THP-1 and U937. Cholesterol is a lipid molecule with very low water solubility, making it difficult to establish a specific trafficking route or to demonstrate the functionality of free cholesterol in cells. In the present study, all hypercholesterolaemia patients included were characterized according to a total cholesterol of ≥240 mg/dl (6.22 mmol/l), which is considered to be the high-risk serum lipid concentration [28]. For imitating the clinical conditions better, we used a previously established cholesterol-loading model that has properties similar to or identical with those of simple diffusion. THP-1 and U937 cells were treated with cholesterol (dissolved in 100% ethanol and added to the culture medium) as in earlier studies [30,31], and no obvious apoptosis was detected (Supplementary Figures S3A and S3B). Although the intracellular distribution of loaded cholesterol is unknown, compared with vehicle control, cholesterol loading indeed increased the total cellular cholesterol level by 80%, and up-regulated the expression of NR4A1.

Hypercholesterolaemia can affect epigenetic patterning. In an in vivo model, Alkemade et al. [10] demonstrated that both in utero programming and diet-induced hypercholesterolaemia change histone methylation and expression of accompanying lysine methyltransferases in vascular ECs (endothelial cells) and smooth muscle cells. LDL-C up-regulates p66shc expression via inducing histone H3 hyperacetylation and DNA hypomethylation in the promoter region of p66shc in human ECs [11]. In blood from patients with familial hypercholesterolaemia as well, DNA hypomethylation of the ABCG1 (ATP-binding cassette transporter G1) and ADRB3 (β-adrenoreceptor 3) gene promoters is significantly associated with plasma LDL-C level [12]. Similar to these results, we find that high cholesterol stimulates NR4A1 transcriptional activation through enhancing p300 and decreasing HDAC7 recruitment to the NR4A1 promoter region, resulting in histone H3 hyperacetylation in monocytes. We did not observe the effects of cholesterol on histone H3 acetylation and mRNA expression of DHRS4 (dehydrogenase/reductase SDR family 4) (Supplementary Figure S6), an unrelated gene which we reported to be regulated by acetylation of histone H3 in our previous study [32]. Thus we think that NR4A1 expression is affected directly by hypercholesterolaemia, and cholesterol-induced histone H3 hyperacetylation is a specific response in NR4A1 expression. Together with other studies [18,42,43], our findings suggest that stimulation of the NR4A1 promoter induces recruitment of RNA pol II and inhibits HDAC activity in order to stimulate transcription of NR4A1. Cholesterol treatment results in p300 binding to the NR4A1 promoter to oppose HDAC (HDAC7) activity and allow initiation of NR4A1 transcription in monocytes. Such a mechanism of derepression by recruitment of HAT to counteract HDAC is a common mechanism for cessation of transcriptional repression by HDAC [44,45]. Furthermore, we also identified the DNA methylation sites in the NR4A1 promoter region through bioinformatic analysis (Supplementary Figure S7A). Further study revealed that the DNA methylation levels in the NR4A1 promoter are very low both in hypercholesterolaemia patients and healthy individuals, decreasing from 1.2% in the healthy controls to 0.3% in patients (Supplementary Figures S7B and S7C). Therefore histone H3 acetylation plays a major role in epigenetic regulation of NR4A1 by hypercholesterolaemia.

Following this, we investigated the role of NR4A1 in high cholesterol-induced inflammation. There is increasing evidence that NR4A1 regulates inflammatory processes in atherosclerosis. Indeed, NR4A1 is highly expressed in macrophages in human atherosclerotic lesions, and inhibits expression and secretion of several pro-inflammatory cytokines and chemokines, such as IL-1β, IL-6, IL-8 and MCP-1 [14,2022]. In monocytes and macrophages, deletion of NR4A1 enhances Toll-like receptor expression and NF-κB activity, results in macrophage polarization towards the pro-inflammatory phenotype and increases atherosclerosis [2124]. NR4A1 has been also reported to be up-regulated by oxLDL (oxidized low-density lipoprotein) through the p38 MAPK (mitogen-activated protein kinase) signal pathway in murine macrophages [39], by VEGF (vascular endothelial growth factor) through PKD (protein kinase D)/HDAC7/MEF2 (myocyte enhancer factor 2) and ERK1/2 (extracellular-signal-regulated kinase 1/2) pathways in ECs [46], and subsequently suppressed inflammatory response induced by these stimuli. Those results indicate that NR4A1 expression is induced in response to some inflammatory stimuli and is regulated by different pathways. Therefore NR4A1 may be a part of the anti-inflammatory response that accompanies inflammation. Similarly, the present study also shows that NR4A1 exerts a strong inhibitory effect on expression and secretion of MCP-1 and IL-6 both at the basal level and in response to cholesterol in monocytes. This view is drawn from the results from both loss-of-function (siRNA oligonucleotides) and gain-of-function (agonist CsnB) experiments. This is underscored by experimental findings showing the effect of NR4A1 on inflammatory cytokines in THP-1 macrophage-derived foam cells and apoE−/− (apolipoprotein E) mice fed on a high-fat/high-cholesterol diet [47].

The NF-κB signalling pathway is known to play an important role in inflammation. Previous studies have identified NR4A1 as a potent inhibitor of NF-κB in inflammatory response. Ismail et al. [46] and You et al. [34] reported that, in ECs, NR4A1 inhibits NF-κB activation through up-regulation of IκBα (inhibitor of NF-κB α), by binding to an NR4A1-binding element in the IκBα promoter [34,46]. Another mechanism by which NR4A1 influences NF-κB activation is through direct interaction with the p65 subunit of NF-κB, and blocking p65 binding to its κB element to attenuate the production of pro-inflammatory cytokines [48,49]. These studies suggest that the orphan nuclear receptor NR4A1 is induced by inflammatory stimuli, and exerts an anti-inflammatory effects by suppressing NF-κB activity. These findings could explain why NR4A1 inhibition in studies of ours and others [2123] obviously promotes an inflammatory response. Therefore the regulation, expression and activity of NR4A1 represent a potential target for the prevention and treatment of inflammatory diseases.

In summary, the present study confirms for the first time that, in monocytes, hypercholesterolaemia could induce chronic low-grade inflammation, and at the same time induce NR4A1 expression through enhancing histone acetylation within the NR4A1 promoter. NR4A1, as a self-protective mechanism, attenuates expression and secretion of pro-inflammatory cytokines, maintains the balance between pro-inflammatory and anti-inflammatory pathways, and protects against the hypercholesterolaemia-induced inflammatory response.

AUTHOR CONTRIBUTION

Xina Xie, Bin Liang and Dongyang Huang designed the research. Xina Xie, Xuhong Song, Song Yuan and Haitao Cai performed the experiments. Xina Xie, Yequn Chen and Xiaolan Chang contributed patient blood samples and case selection. Xina Xie analysed the data. Xina Xie, Bin Liang and Dongyang Huang wrote the paper.

We thank Stanley L. Lin for a critical reading of the paper before submission and acknowledge Yiteng Huang for help with collecting blood samples.

FUNDING

This work was supported by the National Natural Science Foundation of China (NSFC) [grant numbers 31371333 and 81400616], and Guangdong Natural Science Foundation [grant number S2012030006289].

Abbreviations

     
  • Ace-H3

    acetylated histone H3

  •  
  • BMI

    body mass index

  •  
  • CRP

    C-reactive protein

  •  
  • CsnB

    cytosporone B

  •  
  • EC

    endothelial cell

  •  
  • HAT

    histone acetyltransferase

  •  
  • HDAC

    histone deacetylase

  •  
  • HDL-C

    high-density lipoprotein cholesterol

  •  
  • IκBα

    inhibitor of NF-κB α

  •  
  • IL

    interleukin

  •  
  • LDL-C

    low-density lipoprotein cholesterol

  •  
  • MCP-1

    monocyte chemoattractant protein 1

  •  
  • NF-κB

    nuclear factor κB

  •  
  • NR

    nuclear receptor

  •  
  • pol

    polymerase

  •  
  • qRT-PCR

    quantitative real-time reverse transcription–PCR

  •  
  • RBC

    red blood cell

  •  
  • TRIG

    triacylglycerol

  •  
  • WBC

    white blood cell

References

References
1
Charakida
 
M.
Tousoulis
 
D.
Skoumas
 
I.
Pitsavos
 
C.
Vasiliadou
 
C.
Stefanadi
 
E.
Antoniades
 
C.
Latsios
 
G.
Siasos
 
G.
Stefanadis
 
C.
 
Inflammatory and thrombotic processes are associated with vascular dysfunction in children with familial hypercholesterolemia
Atherosclerosis
2009
, vol. 
204
 (pg. 
532
-
537
)
[PubMed]
2
Wilson
 
P.W.
Nam
 
B.H.
Pencina
 
M.
D’Agostino
 
R.B., Sr.
Benjamin
 
E.J.
O’Donnell
 
C.J.
 
C-reactive protein and risk of cardiovascular disease in men and women from the Framingham Heart Study
Arch. Intern. Med.
2005
, vol. 
165
 (pg. 
2473
-
2478
)
[PubMed]
3
Fontes
 
J.D.
Yamamoto
 
J.F.
Larson
 
M.G.
Wang
 
N.
Dallmeier
 
D.
Rienstra
 
M.
Schnabel
 
R.B.
Vasan
 
R.S.
Keaney
 
J.F.
Benjamin
 
E.J.
 
Clinical correlates of change in inflammatory biomarkers: the Framingham Heart Study
Atherosclerosis
2013
, vol. 
228
 (pg. 
217
-
223
)
[PubMed]
4
Pedersen
 
T.R.
Assmann
 
G.
Bassand
 
J.-P.
Chapman
 
M.J.
Erbel
 
R.
Sirtori
 
C.
 
Reducing residual cardiovascular risk: the relevance of raising high-density lipoprotein cholesterol in patients on cholesterol-lowering treatment
Diab. Vasc. Dis. Res.
2006
, vol. 
3
 (pg. 
S1
-
S12
)
5
Hovland
 
A.
Aagnes
 
I.
Brekke
 
O.L.
Flage
 
J.H.
Lappegard
 
K.T.
 
No evidence of impaired endothelial function or altered inflammatory state in patients with familial hypercholesterolemia treated with statins
J. Clin. Lipidol.
2010
, vol. 
4
 (pg. 
288
-
292
)
[PubMed]
6
Baigent
 
C.
Keech
 
A.
Kearney
 
P.M.
Blackwell
 
L.
Buck
 
G.
Pollicino
 
C.
Kirby
 
A.
Sourjina
 
T.
Peto
 
R.
Collins
 
R.
, et al 
Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins
Lancet
2005
, vol. 
366
 (pg. 
1267
-
1278
)
[PubMed]
7
Brown
 
J.D.
Plutzky
 
J.
 
Peroxisome proliferator-activated receptors as transcriptional nodal points and therapeutic targets
Circulation
2007
, vol. 
115
 (pg. 
518
-
533
)
[PubMed]
8
Bensinger
 
S.J.
Tontonoz
 
P.
 
Integration of metabolism and inflammation by lipid-activated nuclear receptors
Nature
2008
, vol. 
454
 (pg. 
470
-
477
)
[PubMed]
9
Pols
 
T.W.
Bonta
 
P.I.
de Vries
 
C.J.
 
NR4A nuclear orphan receptors: protective in vascular disease
Curr. Opin. Lipidol.
2007
, vol. 
18
 (pg. 
515
-
520
)
[PubMed]
10
Alkemade
 
F.E.
van Vliet
 
P.
Henneman
 
P.
van Dijk
 
K.W.
Hierck
 
B.P.
van Munsteren
 
J.C.
Scheerman
 
J.A.
Goeman
 
J.J.
Havekes
 
L.M.
Gittenberger-de Groot
 
A.C.
, et al 
Prenatal exposure to apoE deficiency and postnatal hypercholesterolemia are associated with altered cell-specific lysine methyltransferase and histone methylation patterns in the vasculature
Am. J. Pathol.
2010
, vol. 
176
 (pg. 
542
-
548
)
[PubMed]
11
Kim
 
Y.R.
Kim
 
C.S.
Naqvi
 
A.
Kumar
 
A.
Kumar
 
S.
Hoffman
 
T.A.
Irani
 
K.
 
Epigenetic upregulation of p66shc mediates low-density lipoprotein cholesterol-induced endothelial cell dysfunction
Am. J. Physiol. Heart Circ. Physiol.
2012
, vol. 
303
 (pg. 
H189
-
H196
)
[PubMed]
12
Guay
 
S.P.
Brisson
 
D.
Lamarche
 
B.
Biron
 
S.
Lescelleur
 
O.
Biertho
 
L.
Marceau
 
S.
Vohl
 
M.C.
Gaudet
 
D.
Bouchard
 
L.
 
ADRB3 gene promoter DNA methylation in blood and visceral adipose tissue is associated with metabolic disturbances in men
Epigenomics
2014
, vol. 
6
 (pg. 
33
-
43
)
[PubMed]
13
Guay
 
S.P.
Brisson
 
D.
Lamarche
 
B.
Gaudet
 
D.
Bouchard
 
L.
 
Epipolymorphisms within lipoprotein genes contribute independently to plasma lipid levels in familial hypercholesterolemia
Epigenetics
2014
, vol. 
9
 (pg. 
718
-
729
)
[PubMed]
14
Hamers
 
A.A.
Hanna
 
R.N.
Nowyhed
 
H.
Hedrick
 
C.C.
de Vries
 
C.J.
 
NR4A nuclear receptors in immunity and atherosclerosis
Curr. Opin. Lipidol.
2013
, vol. 
24
 (pg. 
381
-
385
)
[PubMed]
15
Zhao
 
Y.
Bruemmer
 
D.
 
NR4A orphan nuclear receptors: transcriptional regulators of gene expression in metabolism and vascular biology
Arterioscler. Thromb. Vasc. Biol.
2010
, vol. 
30
 (pg. 
1535
-
1541
)
[PubMed]
16
van Tiel
 
C.M.
Kurakula
 
K.
Koenis
 
D.S.
van der Wal
 
E.
de Vries
 
C.J.
 
Dual function of Pin1 in NR4A nuclear receptor activation: enhanced activity of NR4As and increased Nur77 protein stability
Biochim. Biophys. Acta
2012
, vol. 
1823
 (pg. 
1894
-
1904
)
[PubMed]
17
Kang
 
S.A.
Na
 
H.
Kang
 
H.J.
Kim
 
S.H.
Lee
 
M.H.
Lee
 
M.O.
 
Regulation of Nur77 protein turnover through acetylation and deacetylation induced by p300 and HDAC1
Biochem. Pharmacol.
2010
, vol. 
80
 (pg. 
867
-
873
)
[PubMed]
18
Hawk
 
J.D.
Bookout
 
A.L.
Poplawski
 
S.G.
Bridi
 
M.
Rao
 
A.J.
Sulewski
 
M.E.
Kroener
 
B.T.
Manglesdorf
 
D.J.
Abel
 
T.
 
NR4A nuclear receptors support memory enhancement by histone deacetylase inhibitors
J. Clin. Invest.
2012
, vol. 
122
 (pg. 
3593
-
3602
)
[PubMed]
19
Dequiedt
 
F.
Kasler
 
H.
Fischle
 
W.
Kiermer
 
V.
Weinstein
 
M.
Herndier
 
B.G.
Verdin
 
E.
 
HDAC7, a thymus-specific class II histone deacetylase, regulates Nur77 transcription and TCR-mediated apoptosis
Immunity
2003
, vol. 
18
 (pg. 
687
-
698
)
[PubMed]
20
Bonta
 
P.I.
van Tiel
 
C.M.
Vos
 
M.
Pols
 
T.W.
van Thienen
 
J.V.
Ferreira
 
V.
Arkenbout
 
E.K.
Seppen
 
J.
Spek
 
C.A.
van der Poll
 
T.
, et al 
Nuclear receptors Nur77, Nurr1, and NOR-1 expressed in atherosclerotic lesion macrophages reduce lipid loading and inflammatory responses
Arterioscler. Thromb. Vasc. Biol.
2006
, vol. 
26
 (pg. 
2288
-
2294
)
[PubMed]
21
Hanna
 
R.N.
Shaked
 
I.
Hubbeling
 
H.G.
Punt
 
J.A.
Wu
 
R.
Herrley
 
E.
Zaugg
 
C.
Pei
 
H.
Geissmann
 
F.
Ley
 
K.
Hedrick
 
C.C.
 
NR4A1 (Nur77) deletion polarizes macrophages toward an inflammatory phenotype and increases atherosclerosis
Circ. Res.
2012
, vol. 
110
 (pg. 
416
-
427
)
[PubMed]
22
Hamers
 
A.A.
Vos
 
M.
Rassam
 
F.
Marinkovic
 
G.
Kurakula
 
K.
van Gorp
 
P.J.
de Winther
 
M.P.
Gijbels
 
M.J.
de Waard
 
V.
de Vries
 
C.J.
 
Bone marrow-specific deficiency of nuclear receptor Nur77 enhances atherosclerosis
Circ. Res.
2012
, vol. 
110
 (pg. 
428
-
438
)
[PubMed]
23
Hilgendorf
 
I.
Gerhardt
 
L.M.
Tan
 
T.C.
Winter
 
C.
Holderried
 
T.A.
Chousterman
 
B.G.
Iwamoto
 
Y.
Liao
 
R.
Zirlik
 
A.
Scherer-Crosbie
 
M.
, et al 
Ly-6Chigh monocytes depend on Nr4a1 to balance both inflammatory and reparative phases in the infarcted myocardium
Circ. Res.
2014
, vol. 
114
 (pg. 
1611
-
1622
)
[PubMed]
24
Hanna
 
R.N.
Carlin
 
L.M.
Hubbeling
 
H.G.
Nackiewicz
 
D.
Green
 
A.M.
Punt
 
J.A.
Geissmann
 
F.
Hedrick
 
C.C.
 
The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C− monocytes
Nat. Immunol.
2011
, vol. 
12
 (pg. 
778
-
785
)
[PubMed]
25
Geissmann
 
F.
Manz
 
M.G.
Jung
 
S.
Sieweke
 
M.H.
Merad
 
M.
Ley
 
K.
 
Development of monocytes, macrophages, and dendritic cells
Science
2010
, vol. 
327
 (pg. 
656
-
661
)
[PubMed]
26
Perez-Sieira
 
S.
Lopez
 
M.
Nogueiras
 
R.
Tovar
 
S.
 
Regulation of NR4A by nutritional status, gender, postnatal development and hormonal deficiency
Sci. Rep.
2014
, vol. 
4
 pg. 
4264
 
[PubMed]
27
Veum
 
V.L.
Dankel
 
S.N.
Gjerde
 
J.
Nielsen
 
H.J.
Solsvik
 
M.H.
Haugen
 
C.
Christensen
 
B.J.
Hoang
 
T.
Fadnes
 
D.J.
Busch
 
C.
, et al 
The nuclear receptors NUR77, NURR1 and NOR1 in obesity and during fat loss
Int. J. Obes.
2012
, vol. 
36
 (pg. 
1195
-
1202
)
28
Jellinger
 
P.S.
Smith
 
D.A.
Mehta
 
A.E.
Ganda
 
O.
Handelsman
 
Y.
Rodbard
 
H.W.
Shepherd
 
M.D.
Seibel
 
J.A.
 
American Association of Clinical Endocrinologists’ Guidelines for Management of Dyslipidemia and Prevention of Atherosclerosis
Endocr. Pract.
2012
, vol. 
18
 
Suppl. 1
(pg. 
1
-
78
)
29
Peterson
 
T.E.
Poppa
 
V.
Ueba
 
H.
Wu
 
A.
Yan
 
C.
Berk
 
B.C.
 
Opposing effects of reactive oxygen species and cholesterol on endothelial nitric oxide synthase and endothelial cell caveolae
Circ. Res.
1999
, vol. 
85
 (pg. 
29
-
37
)
[PubMed]
30
Wang
 
T.
Chen
 
Z.
Wang
 
X.
Shyy
 
J.Y.
Zhu
 
Y.
 
Cholesterol loading increases the translocation of ATP synthase β chain into membrane caveolae in vascular endothelial cells
Biochim. Biophys. Acta
2006
, vol. 
1761
 (pg. 
1182
-
1190
)
[PubMed]
31
Fu
 
C.
He
 
J.
Li
 
C.
Shyy
 
J.Y.
Zhu
 
Y.
 
Cholesterol increases adhesion of monocytes to endothelium by moving adhesion molecules out of caveolae
Biochim. Biophys. Acta
2010
, vol. 
1801
 (pg. 
702
-
710
)
[PubMed]
32
Li
 
Q.
Su
 
Z.
Xu
 
X.
Liu
 
G.
Song
 
X.
Wang
 
R.
Sui
 
X.
Liu
 
T.
Chang
 
X.
Huang
 
D.
 
AS1DHRS4, a head-to-head natural antisense transcript, silences the DHRS4 gene cluster in cis and trans
Proc. Natl. Acad. Sci. U.S.A.
2012
, vol. 
109
 (pg. 
14110
-
14115
)
[PubMed]
33
Schneider
 
R.
Bannister
 
A.J.
Myers
 
F.A.
Thorne
 
A.W.
Crane-Robinson
 
C.
Kouzarides
 
T.
 
Histone H3 lysine 4 methylation patterns in higher eukaryotic genes
Nat. Cell Biol.
2004
, vol. 
6
 (pg. 
73
-
77
)
[PubMed]
34
You
 
B.
Jiang
 
Y.Y.
Chen
 
S.
Yan
 
G.
Sun
 
J.
 
The orphan nuclear receptor Nur77 suppresses endothelial cell activation through induction of IκBα expression
Circ. Res.
2009
, vol. 
104
 (pg. 
742
-
749
)
[PubMed]
35
Zhan
 
Y.
Du
 
X.
Chen
 
H.
Liu
 
J.
Zhao
 
B.
Huang
 
D.
Li
 
G.
Xu
 
Q.
Zhang
 
M.
Weimer
 
B.C.
, et al 
Cytosporone B is an agonist for nuclear orphan receptor Nur77
Nat. Chem. Biol.
2008
, vol. 
4
 (pg. 
548
-
556
)
[PubMed]
36
Frolov
 
A.
Hui
 
D.Y.
 
The modern art of atherosclerosis: a picture of colorful plants, cholesterol, and inflammation
Arterioscler. Thromb. Vasc. Biol.
2007
, vol. 
27
 (pg. 
450
-
452
)
[PubMed]
37
Pei
 
L.
Waki
 
H.
Vaitheesvaran
 
B.
Wilpitz
 
D.C.
Kurland
 
I.J.
Tontonoz
 
P.
 
NR4A orphan nuclear receptors are transcriptional regulators of hepatic glucose metabolism
Nat. Med.
2006
, vol. 
12
 (pg. 
1048
-
1055
)
[PubMed]
38
Huang
 
Q.
Xue
 
J.
Zou
 
R.
Cai
 
L.
Chen
 
J.
Sun
 
L.
Dai
 
Z.
Yang
 
F.
Xu
 
Y.
 
NR4A1 is associated with chronic low-grade inflammation in patients with type 2 diabetes
Exp. Ther. Med.
2014
, vol. 
8
 (pg. 
1648
-
1654
)
[PubMed]
39
Shao
 
Q.
Shen
 
L.H.
Hu
 
L.H.
Pu
 
J.
Qi
 
M.Y.
Li
 
W.Q.
Tian
 
F.J.
Jing
 
Q.
He
 
B.
 
Nuclear receptor Nur77 suppresses inflammatory response dependent on COX-2 in macrophages induced by oxLDL
J. Mol. Cell. Cardiol.
2010
, vol. 
49
 (pg. 
304
-
311
)
[PubMed]
40
Busnelli
 
M.
Manzini
 
S.
Froio
 
A.
Vargiolu
 
A.
Cerrito
 
M.G.
Smolenski
 
R.T.
Giunti
 
M.
Cinti
 
A.
Zannoni
 
A.
Leone
 
B.E.
, et al 
Diet induced mild hypercholesterolemia in pigs: local and systemic inflammation, effects on vascular injury: rescue by high-dose statin treatment
PLoS One
2013
, vol. 
8
 pg. 
e80588
 
[PubMed]
41
Ruggieri
 
A.
Gambardella
 
L.
Maselli
 
A.
Vona
 
R.
Anticoli
 
S.
Panusa
 
A.
Malorni
 
W.
Matarrese
 
P.
 
Statin-induced impairment of monocyte migration is gender-related
J. Cell. Physiol.
2014
, vol. 
229
 (pg. 
1990
-
1998
)
[PubMed]
42
Fass
 
D.M.
Butler
 
J.E.
Goodman
 
R.H.
 
Deacetylase activity is required for cAMP activation of a subset of CREB target genes
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
43014
-
43019
)
[PubMed]
43
Wilson
 
A.J.
Chueh
 
A.C.
Togel
 
L.
Corner
 
G.A.
Ahmed
 
N.
Goel
 
S.
Byun
 
D.S.
Nasser
 
S.
Houston
 
M.A.
Jhawer
 
M.
, et al 
Apoptotic sensitivity of colon cancer cells to histone deacetylase inhibitors is mediated by an Sp1/Sp3-activated transcriptional program involving immediate-early gene induction
Cancer Res.
2010
, vol. 
70
 (pg. 
609
-
620
)
[PubMed]
44
Steffan
 
J.S.
Bodai
 
L.
Pallos
 
J.
Poelman
 
M.
McCampbell
 
A.
Apostol
 
B.L.
Kazantsev
 
A.
Schmidt
 
E.
Zhu
 
Y.Z.
Greenwald
 
M.
, et al 
Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila
Nature
2001
, vol. 
413
 pg. 
739- 743
 
[PubMed]
45
Jin
 
S.
Scotto
 
K.W.
 
Transcriptional regulation of the MDR1 gene by histone acetyltransferase and deacetylase is mediated by NF-Y
Mol. Cell. Biol.
1998
, vol. 
18
 (pg. 
4377
-
4384
)
[PubMed]
46
Ismail
 
H.
Mofarrahi
 
M.
Echavarria
 
R.
Harel
 
S.
Verdin
 
E.
Lim
 
H.W.
Jin
 
Z.G.
Sun
 
J.
Zeng
 
H.
Hussain
 
S.N.
 
Angiopoietin-1 and vascular endothelial growth factor regulation of leukocyte adhesion to endothelial cells: role of nuclear receptor-77
Arterioscler. Thromb. Vasc. Biol.
2012
, vol. 
32
 (pg. 
1707
-
1716
)
[PubMed]
47
Hu
 
Y.W.
Zhang
 
P.
Yang
 
J.Y.
Huang
 
J.L.
Ma
 
X.
Li
 
S.F.
Zhao
 
J.Y.
Hu
 
Y.R.
Wang
 
Y.C.
Gao
 
J.J.
, et al 
Nur77 decreases atherosclerosis progression in apoE−/− mice fed a high-fat/high-cholesterol diet
PLoS One
2014
, vol. 
9
 pg. 
e87313
 
[PubMed]
48
Hong
 
C.Y.
Park
 
J.H.
Ahn
 
R.S.
Im
 
S.Y.
Choi
 
H.S.
Soh
 
J.
Mellon
 
S.H.
Lee
 
K.
 
Molecular mechanism of suppression of testicular steroidogenesis by proinflammatory cytokine tumor necrosis factor alpha
Mol. Cell. Biol.
2004
, vol. 
24
 (pg. 
2593
-
2604
)
[PubMed]
49
Li
 
L.
Liu
 
Y.
Chen
 
H.Z.
Li
 
F.W.
Wu
 
J.F.
Zhang
 
H.K.
He
 
J.P.
Xing
 
Y.Z.
Chen
 
Y.
Wang
 
W.J.
, et al 
Impeding the interaction between Nur77 and p38 reduces LPS-induced inflammation
Nat. Chem. Biol.
2015
, vol. 
11
 (pg. 
339
-
346
)
[PubMed]

Supplementary data