Published online before print July 27, 2006, doi: 10.1161/01.ATV.0000238346.84458.5d
© 2006 American Heart Association, Inc.
Vascular Biology |
Nuclear Receptors Nur77, Nurr1, and NOR-1 Expressed in Atherosclerotic Lesion Macrophages Reduce Lipid Loading and Inflammatory Responses
From the Departments of Medical Biochemistry (P.I.B., C.M.v.T., M.V., J.V.v.T., V.F., E.K.A., H.P., C.J.M.d.V.), Experimental Internal Medicine (C.A.S., T.v.d.P.), and the AMC Liver Center (J.S.), Academic Medical Center, University of Amsterdam, The Netherlands.
Correspondence to Dr. Carlie J. de Vries, Department of Medical Biochemistry K1–116, Academic Medical Center, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands. E-mail C.J.deVries{at}amc.uva.nl
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Abstract |
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Objective— Atherosclerosis is an inflammatory disease in which macrophage activation and lipid loading play a crucial role. In this study, we investigated expression and function of the NR4A nuclear receptor family, comprising Nur77 (NR4A1, TR3), Nurr1 (NR4A2), and NOR-1 (NR4A3) in human macrophages.
Methods and Results— Nur77, Nurr1, and NOR-1 are expressed in early and advanced human atherosclerotic lesion macrophages primarily in areas of plaque activation/progression as detected by in situ-hybridization and immunohistochemistry. Protein expression localizes to the nucleus. Primary and THP-1 macrophages transiently express NR4A-factors in response to lipopolysaccharide and tumor necrosis factor 
. Lentiviral overexpression of Nur77, Nurr1, or NOR-1 reduces expression and production of interleukin (IL)-1ß and IL-6 proinflammatory cytokines and IL-8, macrophage inflammatory protein-1 and -1ß and monocyte chemoattractant protein-1 chemokines. In addition, NR4A-factors reduce oxidized–low-density lipoprotein uptake, consistent with downregulation of scavenger receptor-A, CD36, and CD11b macrophage marker genes. Knockdown of Nur77 or NOR-1 with gene-specific lentiviral short-hairpin RNAs resulted in enhanced cytokine and chemokine synthesis, increased lipid loading, and augmented CD11b expression, demonstrating endogenous NR4A-factors to inhibit macrophage activation, foam-cell formation, and differentiation.
Conclusion— NR4A-factors are expressed in human atherosclerotic lesion macrophages and reduce human macrophage lipid loading and inflammatory responses, providing further evidence for a protective role of NR4A-factors in atherogenesis.
We demonstrate that the NR4A family of transcription factors, comprising Nur77, Nurr1, and NOR-1, is expressed in human atherosclerotic lesion macrophages and reduces inflammatory responses and lipid loading involving inhibition of macrophage differentiation. Consequently, these receptors reduce foam-cell formation and may prevent lesion macrophages from producing excessive chemokines and cytokines.
Key Words: atherosclerosis • foam-cell formation • inflammation • macrophage function • nuclear orphan receptors • NR4A
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Introduction |
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Atherosclerosis is a chronic inflammatory disease involving deregulation of both the immune system and lipid metabolism.1,2 Macrophages, imperative in the innate immune system, are involved in the initiation, progression, and rupture of atherosclerotic lesions as well as in the initiation of smooth muscle cell (SMC)-rich pathologies like restenosis.3,4 At the onset of atherosclerosis, monocytes are locally recruited to the arterial vessel wall, where these cells differentiate into macrophages. These intimal macrophages ingest modified lipid particles and become lipid-laden foam cells that form a so-called fatty streak. In advanced atherosclerotic lesions, macrophages are localized primarily around a central lipid core and at the shoulder region of the plaque. At the latter site, which is known to be prone to rupture, these cells may be involved in destabilization of the lesion.5 Throughout the progression of atherosclerosis, macrophages produce proinflammatory cytokines, chemokines, growth factors, and matrix-degrading enzymes and are consequently crucial in the chronic inflammatory process in the diseased vessel wall.6,7 Detailed knowledge on the molecular mechanisms involved in the inflammatory and metabolic processes in macrophages is essential to develop novel drug therapies against atherosclerosis. We hypothesized that NR4A nuclear receptors are key regulatory factors involved in modulation of these specific processes in macrophages.
The NR4A nuclear hormone receptors were first described as early response transcription factors expressed on stimulation by growth factors.8–10 This NR4A subfamily comprises 3 members: notably Nur77 (NR4A1, TR3, NGFI-B, NAK-1), Nurr1 (NR4A2, NOT), and NOR-1 (NR4A3, MINOR).11 Like other nuclear receptors, NR4A-factors contain a central DNA-binding domain, comprising 2 zinc-fingers, that bind the consensus response element NBRE (AAAGGTCA) as monomers and the palindromic NurRE element (TGATATTTX6AAAGTCCA) as homo/heterodimers in promoters of specific target genes.12 Furthermore, nuclear receptors consist of an N-terminal domain mediating transactivation and a C-terminal ligand-binding domain. Specific ligands for the NR4A family of transcription factors have not been identified, classifying them as orphan nuclear receptors.13 At the C-terminal domain, both Nur77 and Nurr1 can heterodimerize with RXRs and mediate retinoid responses.14 The NR4A family members have been shown to be functionally involved in T cell and cancer cell apoptosis15–17 and in dopaminergic differentiation of neurons.18
In searching for genes involved in SMC activation in atherogenesis, we revealed the induction of Nur77 and NOR-1 expression in in vitro–activated SMCs.19 Furthermore, we have shown expression of all 3 NR4A-factors in atherosclerotic lesions and in cultured human SMCs and endothelial cells (ECs).20,21 We demonstrated that Nur77 overexpression in vitro inhibits proliferation of both SMCs and ECs. In vivo overexpression of Nur77 under the control of an arterial SMC-specific promoter in transgenic mice protects against SMC-rich lesion formation.19 Other nuclear receptors, notably PPARs and LXRs, play an important role in both SMC and macrophage function relevant to atherosclerosis and restenosis.22–25 However, the function of NR4A-factors in human macrophages is unknown.
In the current study, we show, for the first time to the best of our knowledge, expression of all 3 NR4A family members Nur77, Nurr1, and NOR-1 in human atherosclerotic lesion macrophages, and we demonstrate that these factors reduce the uptake of oxidized low-density lipoprotein (ox-LDL) as well as the inflammatory response in human macrophages.
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Materials and Methods |
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Details of the Materials and Methods are provided in the online data supplement available at http://atvb.ahajournals.org.
Immunohistochemistry and Double In Situ Immunohistochemistry
Macrophages were detected by Ham56 (DAKO) and SMCs by the antibody directed against SM-actin, 1A4 (DAKO). Anti-Nur77 (M-210), anti-Nurr1 (M-196, Santa Cruz Biotechnology), and anti–NOR-1 (rabbit polyclonal antibody directed against NOR-1 was generated; for data on specificity see Figure I, available online), were used to detect the NR4A-factors. A combination of radioactive gene-specific in situ hybridization and macrophage-specific immunohistochemistry was applied to detect NR4A macrophage-specific expression.20
Lentiviral Vector Construction, Infection, and shRNA Interference
hNur77, hNurr1, hNOR-1, and enhanced green fluorescence protein (EGFP) cDNAs were cloned into the pRRL-cPPt-PGK-PreSIN vector.26 Short hairpin (sh) Nur77and shNOR-1 were cloned into p156RRL-sinPPT-CMV-GFP-PRE/NheI.27 shRNA design and sequences are available in the online data supplement. Virus was produced as described.26 THP-1 and U937 cells were transduced for 24 hours with recombinant lentivirus at a multiplicity of infection (MOI) of 3 and 9, respectively, in the presence of 10 µg/mL DEAE-dextran. After transduction, cells were cultured in suspension for 72 hours, differentiated into macrophages, and cultured as described above. Overexpression of Nur77, Nurr1, NOR-1, and EGFP was verified by flow cytometric analyses (EGFP) and immunofluorescence (Figure II.1, available online). shNur77 and shNOR-1 constructs contained CMV-GFP and transduction efficiency was verified by flow cytometric analysis (GFP). Knockdown was confirmed by RT-PCR (Figure II.2, available online) and immunofluorescence.28
RNA and Protein Analysis
Briefly, RNA was extracted, and cDNA was made. Specific primers for Nur77, Nurr1, NOR-1, scavenger receptor-A (SR-A), CD36, CD11b, interleukin (IL)-1ß, IL-6, IL-8, macrophage inflammatory protein-1 (MIP-1) and-1ß (MIP-1ß), monocyte chemoattractant protein-1 (MCP-1), and ribosomal protein P0 were designed. Primer sequences are available online in the data supplement. All RT-PCR data were corrected for housekeeping gene ribosomal protein P0. Protein levels of IL-1ß, IL-6, and IL-8 were determined in supernatant of cell cultures by BD Cytometric Bead Array according to manufacturers’ protocol.
Lipid Loading, Quantification, and Microscopy
After lentiviral infection THP-1–derived macrophages were treated with DiI-labeled ox-LDL for time periods indicated, subsequently washed twice with PBS, and lysed in isopropanol. After sonification followed by 10 minutes centrifugation (13 000g) DiI-labeled ox-LDL content was measured by fluorometry. For confocal microscopy, cells were cultured on glass and treated with DiI-labeled ox-LDL.
Statistical Analysis
The unpaired Student t test was used to calculate the statistical significance of the expression ratios versus control. P<0.05 was considered statistically significant.
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Nur77, Nurr1, and NOR-1 Are Expressed in Human Atherosclerotic Lesion Macrophages
In previous studies we demonstrated expression of Nur77, Nurr1, and NOR-1 in both SMCs and ECs in atherosclerotic lesions.20,21 In this study, we show expression of Nur77, Nurr1, and NOR-1 in atherosclerotic lesion macrophages by combining macrophage-specific immunostaining with gene-specific in situ hybridization. Aorta specimens of 8 different organ donors (3 males and 5 females, age 40 to 69 years) were characterized by immunohistochemistry according to the American Heart Association guidelines (Table and Figure 1A and 1B).29 The complexity of the lesions analyzed ranged from class II to VI. mRNA expression levels of Nur77, Nurr1, and NOR-1 in lesion macrophages and SMCs were scored, and specific localization of expression in the lesion was indicated. As a typical example of an early lesion, a type II lesion with high mRNA expression levels of all 3 nuclear receptors in macrophages is shown (Figure 1C through 1E;
in the Table). Protein expression of Nur77, Nurr1, and NOR-1 localizes to the nucleus in macrophage-rich areas and is comparable with the mRNA expression pattern (Figure 1F through 1I; 
in the Table). Notably, in complex lesions, prominent macrophage-specific NR4A expression is localized especially to shoulder regions and macrophages infiltrated in the media.
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indicates macrophages; Neo, neointima; Lu, lumen; M, media. Arrows in C, D, and E point at macrophages expressing the specific mRNAs.
Nur77, Nurr1, and NOR-1 Are Expressed in Response to Inflammatory Stimuli and Reduce ox-LDL Lipid Loading
High expression levels of NR4A-factors in atherosclerotic lesion macrophages prompted us to study whether their expression is dependent on inflammatory signaling pathways that are active at diseased areas. In addition, the functional activity of these transcription factors was determined in in vitro studies.
In line with recently published data,30 we observed robust and transient mRNA expression of all 3 NR4A-factors in primary macrophages and in monocytic THP-1 cells in response to lipopolysaccharide (LPS). In addition, we show that NR4A-factors are moderately induced by tumor necrosis factor (TNF) in primary macrophages and highly induced (50- to 150-fold induction) in THP-1 phorbol 12-myristate 13-acetate–maturated macrophages in response to LPS. Immunofluorescent analysis of NOR-1 expression revealed that this protein localizes predominantly to the nucleus after LPS stimulation (Figure III, available online).
To study the function of Nur77, Nurr1, and NOR-1 in macrophages, we infected THP-1 cells with lentiviruses that encode these factors or control Mock-virus and determined the effect on lipid loading, a hallmark of atherosclerosis. Lentiviral overexpression of NR4A nuclear receptors resulted in 80% to 90% transduction efficiency and nuclear localization of the encoded proteins (Figure II.1, available online). Viability of NR4A overexpressing cells was comparable to control cells (data not shown). In macrophages overexpressing NR4A-factors, DiI-labeled ox-LDL uptake was quantified by fluorometry and was shown to be reduced after 3 to 6 hours, with 30% reduction after 24 hours (Figure 2A). Confocal microscopy was performed to assess the cellular localization of DiI-labeled ox-LDL in macrophages. After 24 hours, DiI-fluorescence localizes to lipid vacuoles and fluorescence intensity is relatively low in Nur77-overexpressing macrophages as compared with Mock-lentivirus–infected cells (Figure 2B). Because SR-A and CD36 are important genes involved in modified lipoprotein uptake, mRNA expression levels of these genes were determined by semiquantitative real-time RT-PCR. THP-1 macrophages overexpressing Nur77, Nurr1, or NOR-1 express significantly lower levels of SR-A and CD36 than Mock-virus–infected cells (Figure 2C). In addition, we show that CD11b expression, a general macrophage marker gene, is reduced in both THP-1 and U937 macrophages overexpressing NR4A receptors (Figure 2C and 2D). To unravel the function of endogenous NR4A-factors in foam-cell formation, specific shRNAs against Nur77 and NOR-1 were designed. Knockdown of endogenous Nur77 or NOR-1 resulted in a significant increase in DiI-labeled ox-LDL uptake consistent with a 
2-fold increase in SR-A and CD36 mRNA expression as compared with cells transduced with control shRNA (Figure 3A and 3B). CD11b expression was increased 2.3- and 2.7-fold in shNur77 or shNOR-1 expressing THP-1 macrophages, respectively (Figure 3C).
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Lentiviral Overexpression of Nur77, Nurr1, and NOR-1 Reduces Proinflammatory Cytokine and Chemokine Expression
Next, we assayed NR4A function in cytokine and chemokine synthesis in human THP-1 and U937 macrophages. mRNA levels of proinflammatory cytokines IL-1ß and IL-6 and chemokines IL-8, MIP-1/-ß, and MCP-1 were determined after stimulation with LPS, TNF, or vehicle (Figure 4A). As a control for the activity of LPS and TNF, mRNA levels were assayed in Mock-infected macrophages (Figure 4A). Except for IL-6 expression, which is not detectable in vehicle or TNF-treated cells, mRNA expression levels of these inflammatory genes are induced 20- to 8000-fold by LPS and 3- to 10-fold by TNF. mRNA levels of these chemokines and cytokines analyzed are robustly reduced (2- to 10-fold) in THP-1 macrophages overexpressing either Nur77, Nurr1, or NOR-1 as compared with Mock-infected cells after LPS and TNF stimulation. As an exception, MCP-1 mRNA expression is 2.5-fold induced by TNF in NOR-1 overexpressing macrophages and is not significantly different in Nurr1 overexpressing cells as compared with Mock-infected cells. In addition to the mRNA results described, we determined protein concentrations of IL-1ß, IL-6, and IL-8 (Figure 4B) in the conditioned medium of lentivirus-infected THP-1 macrophages. Conditioned media were collected at 0, 6, and 24 hours after treatment with LPS, and protein concentrations were determined by BD Cytometric Bead Array. Overexpression of Nur77, Nurr1, or NOR-1 results in a significant reduction of LPS-induced secretion of IL-1ß, IL-6, and IL-8 by THP-1 macrophages.
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To provide further evidence for an anti-inflammatory function of NR4A-factors in human macrophages, we analyzed cytokine and chemokine expression in human U937 cells in gain-of-function experiments. After stimulation with LPS, NR4A-factors reduce mRNA expression of IL-8 and MCP-1 substantially as well as IL-6 and IL-8 protein levels in conditioned media of these cells (Figure 4C and 4D).
The inhibiting function of endogenous NR4A-factors in inflammatory responses is substantiated by specific shRNAs against Nur77 or NOR-1. Lentivirally delivered shNur77 or shNOR-1 results in an increase of IL-1ß, IL-8, and MCP-1 mRNA expression after LPS stimulation as compared with control shRNA infected cells (Figure 5A). In addition, Nur77 or NOR-1 knockdown significantly increases IL-1ß and IL-8 protein concentrations in the supernatant of these cells (Figure 5B).
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Discussion |
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Monocyte and macrophage activation together with foam-cell formation are critical events in atherogenesis and other related vascular pathologies. In this study, we demonstrate expression of the NR4A family of nuclear receptors Nur77, Nurr1, and NOR-1 in human atherosclerotic lesion macrophages, especially in areas of plaque activation/progression. So far, colocalization with macrophage marker CD68 has only been reported for Nur77.30 Lentiviral overexpression of NR4A-factors in human macrophages reduced uptake of modified lipid particles substantially as well as expression of proinflammatory cytokines and chemokines. Moreover, shRNA-mediated knockdown of Nur77 or NOR-1 resulted in increased lipid loading and augmented inflammatory responses in these cells, indicating that endogenous NR4A-factors are involved in these processes. A potential mechanism for the effects observed is inhibition of macrophage differentiation, which is consistent with reduced expression of SR-A, CD36, and CD11b in human macrophages in NR4A gain-of-function experiments and enhanced expression of these marker genes in knockdown experiments.
We demonstrated that Nur77, Nurr1, and NOR-1 are transiently induced in response to the inflammatory stimuli LPS and TNF in both primary and THP-1–derived macrophages. LPS especially and, as recently shown, also ox-LDL strongly induce NR4A expression.30 Both LPS and ox-LDL promote Toll-like receptor-4 signaling, which has been shown to be involved in atherogenesis; consequently, these in vitro applied stimuli are relevant to atherosclerosis.31 Paradoxically, NR4A-factors are expressed in areas of plaque progression/activation and are induced by inflammatory stimuli but, as shown in this study, inhibit foam-cell formation and proinflammatory cytokine as well as chemokine production. Similar atheroprotective mechanisms involved in controlling vascular pathologies have been described for other nuclear receptors and are known to be functional during vascular lesion development.20,22–25
Nur77 and NOR-1 have been implicated in apoptosis of T cells involving the transcriptional activity of these transcription factors.15 In macrophages, LPS in combination with the pan-caspase inhibitor zVAD was shown to induce apoptosis involving Nur77, however, the exact mechanism of Nur77 action in zVAD-mediated apoptosis has not been elucidated yet.32 In cancer cells, the apoptotic effect of Nur77 depends on the presence of proapoptotic agents and involves translocation of Nur77 to mitochondria.16 Here, we demonstrate nuclear localization of NR4A proteins in human atherosclerotic lesion macrophages as well as in LPS-stimulated cultured macrophages, suggesting the protein to be predominantly active in this cellular compartment. Furthermore, lentiviral overexpression of NR4A nuclear receptors in both THP-1 and U937 macrophages did not result in a reduced viability of those cells.
The reduced uptake of modified LDL as revealed in this study correlates with downregulation of scavenger receptors SR-A and CD36 expression, which have both been shown to enhance foam cell formation and atherosclerotic lesion size in vivo using macrophage-specific Msr1 and CD36 knockout mice in dedicated atherosclerosis models.33,34 Although there is compelling evidence for a proatherogenic role of these receptors, both in vitro and in vivo, recently this paradigm in atherosclerosis has been challenged.35 The expression of CD36, and a number of other genes, has been shown to be dependent on Nur77 in skeletal muscle.36 It is hypothesized that Nur77 enhances lipolysis in skeletal muscle cells and consequently protects against diet-induced obesity, which supports, in combination with the reduced lipid uptake in macrophages shown, an antiatherogenic function for Nur77-like factors. The underlying mechanism of the tissue-specific regulation of CD36 expression by Nur77 and possibly also its subfamily members NOR-1 and Nurr1 awaits further investigations.
The proinflammatory cytokines and chemokines analyzed in the current study are considered highly relevant for atherogenesis,1–4,6,7 and the NF-
B pathway is vital for expression of these genes.37 In monocytes Nur77 has been isolated in a genome-wide screen that was designed to identify inhibitors of the NF-B pathway.38 It is demonstrated that overexpression of Nur77 in HEK293 cells potently reduces expression of an NF-B reporter construct in response to IL-1ß and TNF. In T cells it was shown that Nur77 is involved in inhibition of NF-B–mediated IL-2 and IL-8 promoter activity, probably through binding of the N-terminal activation domain of Nur77 with the p65 subunit of NF-B,39 comparable to the direct inhibitory interaction of the glucocorticoid receptor with NF-B.40 Taken together, our data may at least in part be explained by transrepression of the NF-B pathway by NR4A-factors. Interestingly, it has recently been shown that overexpression of Nur77 in a mouse macrophage cell-line results in a proinflammatory response involving enhanced expression of inducible I--B kinase (IKKi), an NF-B activating gene.41 The discrepancy may be explained by species difference, because in the latter study the murine promoter of IKKi was shown to contain a functional NBRE, whereas the human IKKi-promoter does not contain this NBRE.
The role of NR4A nuclear receptors in various vascular cell types has been studied in our group and by others.20,21,28,42,43 Nur77 has been shown to promote angiogenesis in dedicated mouse models43 and to inhibit SMC-rich lesion formation in transgenic mice.20 In contrast, inhibition of NOR-1 expression in cultured cells by anti-sense oligonucleotides resulted in reduced SMC and endothelial cell growth, suggesting a stimulatory effect of this NR4A member on proliferation of these vascular cells.42 In the current study, we show that all 3 NR4A family members mediate similar downstream effects in macrophages, except for enhanced MCP-1 expression after TNF stimulation when NOR-1 is overexpressed. A detailed analysis of downstream gene targets for each of these 3 transcription factors will give further insight in gene-specific and cell-specific responses.
In summary, we demonstrate that the NR4A family of transcription factors is expressed in human atherosclerotic lesion macrophages and is functionally involved in inhibition of inflammatory responses and lipid loading. So far, our results point toward atheroprotective properties of NR4A-factors in macrophages and for Nur77 in other vascular cell types as well. Future studies in vitro and in in vivo models will unravel the significance of these transcription factors in atherogenesis and related vascular pathologies.
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Acknowledgments |
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We are grateful to Hella Aberson for her technical assistance.
Source of Funding
This research was supported by the Netherlands Heart Foundation, Molecular Cardiology Program, NHS Grant 93.007.
Disclosures
None.
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Footnotes |
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Original received June 21, 2006; final version accepted July 19, 2006.
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