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Cardiovascular Research 2005 65(3):609-618; doi:10.1016/j.cardiores.2004.10.002
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Copyright © 2004, European Society of Cardiology

The NR4A subfamily of nuclear receptors: new early genes regulated by growth factors in vascular cells

José Martínez-González and Lina Badimon*

Centro de Investigación Cardiovascular, CSIC/ICCC, Hospital de la Santa Creu i Sant Pau, Sant Antoni Maria Claret #167, Barcelona, Spain

* Corresponding author. Tel.: +34 935565882; fax: +34 935565559. Email address: lbadimon{at}csic-iccc.santpau.es

Received 14 July 2004; revised 29 September 2004; accepted 1 October 2004


   Abstract
Top
 Abstract
1. Introduction
 2. The NR4A subfamily...
 3. Regulation of NR4A...
 4. Functional role of...
 5. Concluding remarks
 References
 
The molecular mechanisms regulating endothelial cell activation and vascular smooth muscle cell proliferation are critical in the pathological processes underlying atherosclerosis. Numerous growth factors and cytokines trigger the complex and redundant signaling pathways that regulate cell cycle entry; however, the genes controlling these processes are not fully known. Applying techniques for differential gene expression analysis, new transcription factors have been identified in these mechanisms, among them the three members of the NR4A subfamily of nuclear receptors (NRs). These transcription factors (NOR-1, Nur77 and Nurr1) are products of immediate–early genes whose expression and activity is regulated in a cell-specific manner by a variety of extracellular mitogenic, apoptotic and differentiation stimuli. Unlike most NRs whose transcriptional activity is regulated by direct modulatory ligands, NR4A genes do not appear to require ligand binding for activation, and in vascular cells they are highly responsive to growth factors, cytokines, lipoproteins and thrombin. In this review, we discuss our present knowledge on the role of this subfamily of NRs in vascular cell function.

KEYWORDS Atherosclerosis; Smooth muscle; Receptors; Gene expression; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. The NR4A subfamily...
 3. Regulation of NR4A...
 4. Functional role of...
 5. Concluding remarks
 References
 
The main function of vascular smooth muscle cells (VSMCs) is to maintain vascular tone in response to environmental stimuli. However, during atherogenesis, the increased local secretion of growth factors and cytokines promote VSMC activation and dedifferentiation [1,2]. VSMCs undergo phenotypic changes involving the transient transformation from contractile, resting, fully differentiated cells into proliferative, migratory, dedifferentiated SMCs, which synthesize high amounts of extracellular matrix proteins. This transformation involves the up- or down-regulation of multiple genes coordinately regulated by diverse proteins that control cell cycle entry and other cellular functions such as cell migration and synthetic activity (reviewed in Refs. [3–6]).

In the past few years, new genes have been identified to be involved in atherogenesis, among them different NRs, including peroxisome proliferator-activated receptors (PPARs) and retinoid receptors, such as retinoid X receptor (RXR) and retinoic acid receptor (RAR) [7–9]. NRs comprise a large family of ligand-activated transcription factors that by regulating complex gene programs play critical roles in nearly all aspects of development and adult physiology [10–12]. Currently, ligands have been identified for only half of the known NRs. The remaining receptors, collectively termed orphan NRs, constitute a promising area for research and development since they could be targets for new diagnosis and/or therapeutic strategies.

Recently, we and others have identified Neuron-derived Orphan Receptor-1 (NOR-1) as an early-response gene in VSMC using mRNA-differential display analysis [13–15]. NOR-1, together with Nur77 and Nurr1, form the NR4A (also referred as NGFI-B) subfamily of orphan NRs within the steroid/thyroid receptor superfamily [16]. These transcription factors have been involved in neuroendocrine regulation, neurological disorders, liver regeneration and cancer, and at cellular level they have been implicated in proliferation, differentiation and apoptosis [16]. These genes have emerged as potentially relevant players in the complex network of proteins that regulate endothelial cell activation and VSMC proliferation in inflammation and atherogenesis [13–15,17–22].


   2. The NR4A subfamily of NRs
Top
 Abstract
 1. Introduction
 2. The NR4A subfamily...
3. Regulation of NR4A...
 4. Functional role of...
 5. Concluding remarks
 References
 
2.1. Structure of NRs and the NR4A subfamily
Since the cloning of the estrogen and glucocorticoid receptor as transcription factors in the 1980s, a large number of NRs have been identified. Sequence and functional analysis reveals that these receptors share a modular structure [10–12], characterized by several functional domains: a variable amino-terminal (N-terminal) region (A/B); a central DNA-binding domain (DBD), or region C, and a variable linker region D that connects the DBD to the conserved E/F region in the carboxy-terminus (C-terminus), containing the ligand-binding domain (LBD) (Fig. 1). The N-terminal domain varies in both length and amino acid composition and is responsible for interaction with other transcription factors and for transactivation via its ligand-independent activation function-1 (AF-1) transactivation domain. The DBD comprises two highly conserved zinc finger motifs that mediate specific interaction with DNA. The LBD is essential for the recognition of a small lipophilic molecule (ligand), which ensures both specificity and selectivity of physiological responses, and is thought to be a molecular switch that upon binding shifts the receptor to a transcriptionally active state. This domain may regulate nuclear localization and also contains eight to nine heptad-repeats of hydrophobic amino acids involved in dimerization of NRs. The C-terminal portion of the LBD contains the ligand-dependent AF-2 transactivation domain.


Figure 1
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  		Fig. 1 Structure of a nuclear receptor (A) and schematic representation of the sequences corresponding to NR4A genes (B). Amino acid sequence alignment of NR4A genes and the percent of amino acid identity with the corresponding regions of Nur77 is shown in B.

 
The NR4A subfamily consists of three closely related members: Nur77, also known as nerve growth factor (NGF)-induced clone B (NGFI-B), was the first member of the subfamily identified as a gene induced by NGF in the rat pheocromocytoma cell line PC12 [23]; NOR-1, firstly identified by Ohkura et al. [24] in forebrain neural cells undergoing apoptosis, and Nurr1 (Nur-related factor 1) firstly characterized as a "brain-specific" transcription factor in dopaminergic neurons [25]. The multiple names proposed for these genes by different researchers are summarized in Table 1. These genes share high amino acid identity with other NRs particularly within their DBDs, while the C-terminus exhibits the lowest homology. The genomic structures for NR4A subfamily members are remarkably similar, suggesting that these receptors have evolved from a common ancestral gene [16]. Among the NR4A genes, the most divergent domain is the N-terminal transactivation domain (Fig. 1), which could lead to significant qualitative or quantitative differences among them. There are mRNA variants of the subfamily members, which are generated by alternative splicing [26]. Interestingly, NOR-1 and Nurr1 transcripts encoding C-terminal truncated isoforms, which lack the putative LBD, have been characterized [27–29]. Functional analysis using reporter gene suggest that these truncated forms may act as negative regulators of the NR4A subfamily-target genes because they can bind to the NGFI-B response element (NBRE) preventing transactivation by the full-length isoforms [29].


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  		Table 1 Nomenclature and trivial names for the NR4A genes in different species

 
2.2. Regulation of NR transcriptional activity
The successful identification of ligands for many NRs, including PPARs, the liver X receptors (LXR) and the farnesoid X receptor (FXR), supported the notion that all NRs are regulated by ligand binding [30]. However, recent findings suggest that although members of the NR4A subfamily have structural features of ligand-activated transcription factors, they do not require previous ligand binding for activation [31]. The structure of the Nurr1 LBD determined by X-ray crystallography reveals that it contains no ligand-binding cavity; instead, several tightly packed, bulky hydrophobic residues occupy the space that is available for ligand binding in other NRs. These hydrophobic residues are conserved throughout the NR4A subfamily; thus, the absence of ligand-binding pocket is predicted for all members of this subfamily, suggesting that they are all ligand-independent transcription factors. Similar results exclude the existence of a ligand for DHR38, the orthologue of NOR-1 in Drosophila [32]. In the light of these results, the ligand-independent AF-1 could play a major role mediating transcriptional activation and cofactor recruitment, as recent investigations reveal [33–35].

It was postulated that the ancestral NR was constitutively active [36]; thus, it is not surprising that some NRs could be constitutively active or activated by posttranslational modifications. This could be the case of NR4A genes, because at least Nur77 can be phosphorylated by several kinases, including Akt, ERK2, pp90rsk and c-Jun N-terminal kinase [37–40]. Nur77 phosphorylation state is regulated in response to growth factors such as NGF in neural cells or to vascular endothelial growth factor (VEGF) in endothelial cells (ECs). In particular, phosphorylation in Ser-350 of the DBD by Akt inhibits the DNA binding activity of Nur77 [38,41]. Finally, it has recently been described that sumoylation by PIAS{gamma} represses the transcriptional activation induced by Nurr1 [42].

Therefore, the lack of a "classical" binding site for coactivators seems to rule out regulation of these NRs by naturally occurring ligands. As an "alternative", the transcriptional activity of the NR4A genes is largely regulated by extracellular stimuli, which modulate posttranslational modification processes and determine their mRNA expression levels. The nature of the NR4A subfamily as immediate–early genes induced by multiple signal transduction pathways is a unique feature, which sets these receptors apart from other NRs.


   3. Regulation of NR4A genes by extracellular stimuli in vascular cells
Top
 Abstract
 1. Introduction
 2. The NR4A subfamily...
 3. Regulation of NR4A...
4. Functional role of...
 5. Concluding remarks
 References
 
3.1. Regulation of NR4A genes in VSMC
We and others have recently identified NOR-1, Nur77 and Nurr1 as immediate–early genes strongly induced by growth factors in VSMCs [13–15]. Their respective mRNAs are transiently induced within 1 h, returning to background levels 8 h after induction. In addition, they are overinduced by cycloheximide, a typical feature of immediate–early genes (Fig. 2). NR4A genes seem to be sensitive "markers" of mitogenic stimulus in different cell types. In fact, using array techniques, they have been identified as major responsive genes in ECs exposed to VEGF [21], and in serum-stimulated fibroblasts [43].


Figure 2
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  		Fig. 2 Induction patterns of NOR-1 and Nur77 in VSMC. Northern blots showing time-course induction of NOR-1 and Nur77 by 10% human serum (A); up-regulation of NOR-1 and Nur77 by growth factors and cytokines (5 nmol/l PDGF-BB, 10 U/ml thrombin, 10 nmol/l EGF, 25 nmol/l bFGF, 0.1 nmol/l TGFβ, 5000 U/ml interleukin-1β and 5 nmol/l TNF{alpha}) (B); induction of NOR-1 and Nur77 by phorbol-12-myristate-13-acetate (PMA, PKC activator; 1 and 5 µmol/l), A23187 (Ca2+ ionophore; 5 and 25 µmol/l), 8Br-cAMP (cAMP analogue, activator of PKA; 0.5 and 1 mmol/l) and forskolin (activator of adenylyl cyclase; 10 and 50 µmol/l) (C); inhibition of serum-induced expression of NOR-1 and Nur77 by 1 µg/ml pertussis toxin (PTX, inhibitor of Gi/o proteins), 50 µmol/l PD98059 (inhibitor of MEK1/2), EGTA (calcium chelator; 5 and 10 mmol/l), GF10933X (inhibitor of PKC; 1 and 10 µmol/l) and 2',5'-dideoxyadenosine (DDA, inhibitor of adenylyl cyclase; 0.1 and 0.5 mmol/l). In B–C, cells were induced for 1 h. NI, noninduced cells.

 
Serum and several growth factors commonly involved in atherogenesis, including platelet-derived growth factor (PDGF-BB), epidermal growth factor (EGF) and {alpha}-thrombin significantly induce NOR-1 and Nur77 in human VSMCs (Fig. 2) [15]. In VSMCs, the induction of NOR-1 by serum is significantly higher than that of Nur77. By contrast, Nur77 is more responsive to PDGF-BB and cytokines, either when tested alone (interleukin-1β [IL-1β], tumor necrosis factor-{alpha} [TNF{alpha}]) [15,22] or as a part of the cytokine "cocktail" contained in conditioned media from oxidized LDL-treated human monocytes [13,14]. This cocktail of cytokines also significantly induced Nurr1. Thus, in VSMCs, NR4A genes are induced by mitogenic stimuli signaling through pathways involving tyrosine kinase receptors (TKRs) (i.e., PDGF) and G protein-coupled receptors (GPCRs) (i.e., thrombin).

NOR-1 and Nur77 mRNA levels are induced by compounds that activate specific signaling pathways in VSMCs, such as phorbol-12-myristate-13-acetate (PMA) [a protein kinase C [PKC] activator], A23187 [GenBank] (a calcium ionophore), forskolin (an adenylyl cyclase activator) and the cAMP analogue 8-Br-cAMP (Fig. 2) [15]. However, the strongest inducers were serum and native LDL [15,17], agents that contain bioactive molecules able to activate several cell signaling pathways, and that can enhance gene expression by cross-talk [44–46]. Apart from the well-documented mitogenic effects of serum on VSMCs, LDLs induce vascular cell proliferation acting as "mitogen-like" molecules that increase intracellular free calcium concentration ([Ca2+]i), and activate PKC and mitogen-activated protein kinase (MAPK) pathways (p42/44 MAPK and p38 MAPK) [47,48]. These LDL-induced early intracellular events are independent from the classic LDL receptor but are mediated, at least in a part, by pertussis toxin-sensitive G-proteins [17,47]. Specific inhibitors of any of these pathways significantly interfere with serum- or LDL-induced NOR-1/Nur77 expression (Fig. 2). In particular, PKC and [Ca2+]i, key regulators of VSMC migration and proliferation, are critical for NOR-1 and Nur77 up-regulation in VSMC and for their binding to a consensus NBRE sequence [15]. The redundant network of signaling pathways leading to NOR-1 up-regulation in response to serum mitogens is depicted in Fig. 3.


Figure 3
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  		Fig. 3 Pathways involved in NOR-1 up-regulation in VSMC. Components of serum activate G-protein-coupled receptors (GPCRs) and tyrosine kinase receptors (TKRs). Hydrolysis of phosphatidylinositol-4,5-biphosphate by phospholipase C (PLC) activated by GPCRs or TKRs results in the generation of insitol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). DAG is known to activate protein kinase C (PKC) and IP3 to release Ca2+ from the intracellular stores. PKC could downstream activate Raf and p38 MAPK (p38 in the picture). Adenylyl cyclase (AC) activated by GPCR results in cAMP formation, which activates protein kinase A (PKA). TKRs induce calcium entry from extracellular medium and induce the Ras/Raf/MEK/ERK and p38 MAPK signaling. Increase in [Ca2+]i can activate calcium/calmodulin dependent kinases (Ca/CM). Agents (boxed) that activate (arrows) or inhibit (blocking arrows) NOR-1 expression interfering signaling pathways are indicated. Activators and inhibitors as in Fig. 2 legend. The potential cross-talk among signaling pathways is indicated by shaded arrows. PKA, CA/CM, p38 MAPK and p90RSK are kinases potentially involved in CREB phosphorylation in Ser-133 [59]. Ser-133 phosphorylation promotes transcription of NOR-1 and other target genes by recruitment of the co-activator CREB-binding proteins (CBP), which mediates association with RNA polymerase II complexes. The indicated cross-talk mechanisms and kinases potentially involved in CREB phosphorylation have not been specifically analyzed in relation with NOR up-regulation. [H-89, PKA inhibitor; BAPTA-AM, calcium chelator; SB203580, inhibitor of p38 MAPK)].

 
Depending on the cell type, different transcription factors, including AP-1, NF{kappa}B or MEF-2, participate in the up-regulation of the NR4A genes [49–51]. However, in most tissues and cells, including vascular cells, the transcription of these genes is highly dependent on cAMP response element binding protein (CREB) [15,19,52]. In fact, by microarray using a constitutively active CREB mutant, NR4A genes have been identified as a subset of genes highly responsive to CREB activation [53].

NOR-1 promoter contains three functional CRE sites critical for its transcriptional activation [26]. We have extensively analyzed the role of CREB in the up-regulation of NOR-1 in VSMCs. In these cells, CREB activation seems to be a common output for the signal transduction pathways involved in NOR-1 induction. Stimuli that activate CREB, via phosphorylation in Ser-133, such as serum, thrombin or LDL, strongly induce NOR-1 [15,17]. By contrast, compounds that interfere with signaling pathways upstream of CREB, such as PKC inhibitors or calcium chelators, inhibit CREB phosphoylation and prevent NOR-1 up-regulation. Moreover, cotransfection of VSMCs with a dominant-negative of CREB (CREB with a single mutation in Ser-133), or eliminating CRE sites from the NOR-1 promoter by site-direct mutagenesis completely prevent the transcriptional activity of the NOR-1 promoter [15,17]. Fig. 3 shows pathways that could be involved in CREB phosphorylation [54,55] and in the downstream induction of NOR-1.

Since CREB seems to be a key regulator of NOR-1 in VSMCs and NOR-1 seems to be relevant in VSMC proliferation (see below), one might conclude that CREB activation leads to VSMC proliferation. However, the role of CREB in cell proliferation is certainly ambivalent, because it has been related with both pro- and anti-proliferative status in different cells including VSMCs [54,56–58]. CREB-mediated NOR-1 up-regulation is in agreement with reports that localize activated CREB in the neointima and associate it with VSMC proliferation [57,58], likely as a result of its active role regulating the expression of several cyclins and cyclin-dependent kinases involved in cell cycle progression [59,60].

3.2. Regulation of NR4A genes in endothelial cells
NOR-1 as well as Nur77 and Nurr1 are strikingly induced by growth factors in ECs. Using an Affymetrix oligonucleotide array system, Liu et al. [21] have identified them as the most strongly induced genes in ECs treated with VEGF. The effect of VEGF was mediated by KDR (VEGFR-2), the main receptor involved in the VEGF effect in endothelial cells [61], and was sensitive to p42/44 MAPK, [Ca2+]i, PKC and calcineurin inhibition [21]. By contrast, VEGF-induced expression of NR4A genes was insensitive to rapamycin and to LY2940002, an inhibitor of phosphatidyl-inositol-3-kinase (PI3K) [21]. These authors also found that the signaling mechanisms mediating VEGF regulation of NR4A genes diverge from those responsible for expression of other VEGF-induced genes such as Egr3 at level of calcineurin (a calcium/calmodulin-dependent serine phosphatase). Ca2+/calcineurin regulate cytoplasm to nucleus translocation of the nuclear factor of activated T cells (NFAT). NFAT is a transcription factor that has been associated to Nur77 regulation in T cells [49], and which seems to be critical in Ca2+/calcineurin-dependent vascular cell functions [62]. Interestingly, cyclosporin A (CsA), an immunosuppressor drug that acts as a specific inhibitor of calcineurin and potently blocks angiogenesis [63], inhibited the expression of the three NR4A genes [21]. Thus, CsA would likely prevent NFAT translocation to the nucleus [21] and Nur77 transcriptional activity [64]. Moreover, VEGF not only increases the transcription rate of these genes but concomitantly decreases Nur77 phosphorylation at Ser-350 [21], a negative regulatory site that once phosphorylated inhibits Nur77 transcriptional activity [38,41]. The mechanism of VEGF-induced Nur77 dephosphorylation is unclear but might involve the activation of a serine/threonine phosphatase. Interestingly, Nur77, Nurr1 and NOR-1 have highly homologous DNA-binding domains, including Ser-350 and surrounding residues; therefore, it is likely that all three members are potentially regulated by this mechanism. Fig. 4 shows the pathways be involved in the VEGF-dependent regulation of NR4A genes in ECs.


Figure 4
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  		Fig. 4 Signaling mechanisms mediating VEGF-regulated endothelial expression of NR4A genes. VEGFR-mediated PLC-{gamma} activation (associated with Tyr-1175) and subsequent mobilization of intracellular Ca2+ and activation of PKC and ERK1/2 pathway play key roles in the induction of the expression of NR4A genes by VEGF. Calcium is essential for the activation of calcineurin (a calcium/calmodulin-dependent serine phosphatase) that is inhibited by the immunosuppressive agent cyclosporin A (CsA). Calcineurin activate the nuclear factor of activated T cells (NFAT) (by dephosphorylation) leading to the up-regulation of the three NR4A genes. The VEGF-mediated expression NR4A genes is inhibited by SU5614 (inhibitor of tyrosine kinase activity of VEGFR2) and specific inhibitors of other signaling molecules (BAPTA-AM, GF10933X and U0126). The pathways leading to CREB activation and induction of NOR-1 are also depicted. The role of Akt activation in NR4A activity in endothelial cells is uncertain.

 

   4. Functional role of NR4A genes in vascular cells
Top
 Abstract
 1. Introduction
 2. The NR4A subfamily...
 3. Regulation of NR4A...
 4. Functional role of...
5. Concluding remarks
 References
 
4.1. Role of NR4A genes in cell proliferation
Although NOR-1, Nur77 and Nurr1 expression has been described in human atherosclerotic lesions [13–15], conflicting results have been reported regarding their role in vascular cell proliferation.

We have shown that antisense oligodeoxynucleotides (ODNs) directed against NOR-1 efficiently prevent cell proliferation in serum- or LDL-induced VSMCs [15,17]. Anti-NOR-1 ODNs inhibited cell cycle progression leading to an accumulation of cells in G0/G1 phase, reduced de novo DNA synthesis and efficiently prevented VSMC migration and proliferation in an in vitro model of wound repair [15]. Similarly, these antisense ODNs prevented VEGF- [65] and thrombin-induced (own unpublished results) NOR-1 expression and EC proliferation. Moreover, NOR-1 is strongly induced in porcine coronary arteries subjected to angioplasty (Fig. 5) [15], a mechanical injury process that promote the expression of genes, such as c-fos, c-myc or c-myb, associated with VSMC activation and proliferation [66]. These results are consistent with a role for NOR-1 in the molecular mechanisms underlying VSMC proliferation and accelerated atherosclerosis.


Figure 5
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  		Fig. 5 NOR-1 up-regulation by balloon angioplasty. RT-PCR (A) and in situ RT-PCR (B) showing NOR-1 induction in porcine arteries subjected to percutaneous transluminal coronary angioplasty (PTCA). Injured (a, b) and uninjured (c, d). In situ RT-PCR (a, c) and hematoxylin and eosin stain (b, d). Bar=50 µm. Modified from Ref. [15].

 
For Nur77, distinct results were observed in both VSMCs and ECs [14,20]. Results from transgenic mice models show that Nur77 inhibits formation of VSMC-rich lesions [14]. Transgenic mice expressing either a dominant-negative form of Nur77 (lacking the transactivation domain) or full-length Nur77 under the control of the SM22{alpha} promoter, which direct expression of transgenes specifically to VSMCs, were apparently healthy and no obvious differences were observed in the vasculature between wild-type and transgenic mice. However, when transgenic mice were subjected to carotid artery ligation to induce VSMC proliferation, lesions in mice overexpressing Nur77 were smaller than in wild-type animals. Moreover, mice overexpressing the dominant-negative variant had larger lesions than wild-type mice. Consistent with this, Nur77 also seems to inhibit EC growth [20]. Thus, it is proposed that Nur77 downstream target genes could inhibit the cell cycle. In fact, overexpression of Nur77 in vascular cells increases protein levels of the cyclin-dependent kinase inhibitor p27Kipl and down-regulates cyclin A, [14,20] arresting cells in G1 phase [20]. The expression of p27Kipl correlates inversely with vascular cell proliferation and atherosclerosis [67,68]; however, the mechanism linking Nur77 and p27Kipl is unknown.

Taken together, these data indicate that NOR-1 and Nur77 might be essential for vascular cell activation albeit playing opposite roles. NR4A genes take part in dissimilar functions, including apoptosis, proliferation or cell cycle arrest and differentiation [24,34,35,40,69–72]. In fact, the enhancement of NOR-1 transcriptional activity by 6-mercaptopurine has been related with the antiproliferative effects of this widely used antineoplastic drug [34]. It should be stressed that similar nonspecific roles have been reported for other early genes such as c-fos or c-myc [73,74]. The involvement of NOR-1 in vascular cell proliferation is in agreement with previous results showing that NOR-1 knockout mice exhibit alteration in the proliferation of the semicircular canals of the inner ear [75], and it is essential for early mouse embryogenesis [76]. Similarly, Nur77 seems to regulate mitogenesis in some cancer cell lines [40] but no apparent effects on cell proliferation have been observed in Nur77 null mice. Furthermore, oncogenic transformation has only been described for NOR-1, as a result of its fusion with various N-terminal partners, including EWS, TAF2N and TFG [28,77,78]. The fusion gene products seem to lead to malignant transformation by functioning as transcriptional activators [79].

4.2. Role of NR4A genes in apoptosis
Apoptosis can be mediated through multiple signaling pathways. Mitochondrial release of apoptogenic factors, such as cytochrome c, Smac/DIABLO, and apoptosis-inducing factor [80] can be regulated by caspases, Bcl-2 family proteins, translocation of tumor suppressor p53, and recent findings suggest that also by members of the NR4A subfamily.

In particular, Nur77 is crucial in activation-induced cell death (AICD) of T and B cells [69,81] and in different cancer cell lines [70,82]. Transcription activity and mitochondrial targeting of Nur77 is required for apoptosis [83], and antisense ODNs directed against Nur77 prevent apoptosis [81,82]. Nur77 induction could be a broadly common mechanism of caspase-independent cell death. Indeed, Nur77 participates not only in negative selection of T cells but also in macrophage apoptosis [84]. Although the activation signals leading to cell apoptosis in T cells and macrophages are initiated from different cell surface receptors, one of the common intracellular signaling events seems to be the activation of the ERK pathway and increased activity of MEF2 transcription factor [49,84].

Since the three members of the NR4A subfamily are structurally closely related, they could function redundantly in apoptosis. In fact, Nur77 knockout cannot completely prevent AICD, suggesting that the other NR4A subfamily members may compensate for the absence of Nur77. This role of Nur77 seems to be assumed by Nurr1 in macrophages [84], while NOR-1 and Nur77 demonstrate functional redundancy in an apparently Fas-independent apoptosis in T cells [85]. The overexpression of either Nur77 or NOR-1 in developing T cells of transgenic mice results in massive apoptosis of thymocytes and reduced levels of peripheral T cells [69,85]. Furthermore, NOR-1 is also involved in apoptosis of neural and cancer cells [24,52] and it is one of the major genes induced in peripheral blood mononuclear cells exposed to an apoptotic stimulus as it has been demonstrated by PCR-based suppression subtractive hybridization (SSH) technique [86].

Recently, it was shown that Nur77 could be directly related with key regulators of cell survival and apoptosis. Indeed, phosphorylation of Nur77 on Ser-350 by Akt inhibits T cell death by suppressing the DNA binding activity of Nur77 and stimulating Nur77 association with 14-3-3 [38,41]. In carcinoma cells, Bax is recruited to the mitochondria, secondary to the nucleus-to-cytoplasm translocation of Nur77 [87]. In addition, Nurr77 and Bcl-2 apoptotic machineries seem to be coupled, since Nur77 interacts with the N-terminal loop region of Bcl-2 inducing a conformational change that exposes its BH3 domain, thus, converting Bcl-2 from a anti- to a pro-apoptotic factor [88]. The key role of Akt as a common mediator of cell survival in a variety of circumstances and the central role of Bcl-2 family proteins in apoptosis suggest that NR4A genes could be critical for many other cell types including vascular cells. However, the relevance of NR4A genes in vascular cell apoptosis is largely unexplored. In VSMCs, antioxidants such as pyrrolidinedithiocarbamate (PDTC) induce apoptosis apparently in a Nur77-dependent manner [18]. However, in ECs, Nur77 inhibits cell cycle but does not trigger apoptosis [20]. Future investigations should clarify the role of these genes in the apoptotic/survival processes that regulate vascular wall cellularity.

4.3. Genes regulated by NOR-1, Nur77 and Nurr1
Since these three nuclear receptors are highly homologous, they might be expected to regulate overlapping target genes; however, they exhibit significant differences in their ability to dimerize and to bind to closely related response elements. They bind as monomers to an octamer sequence NBRE, which contains a half site of the canonical hormone response element (HRE) preceded by two additional adenines (AAAGGTCA) [89]. In addition, Nur77 and Nurr1 (but not NOR-1) can heterodimerize with RXR and activate transcription through a DR-5 element in a 9-cis retinoic acid-dependent manner [90,91]. Finally, Nur77, NOR-1 and Nurr1 can bind as homodimers or heterodimers to a Nur77 response element (NurRE), which consists of two inverted NBRE sequences spaced by 6 bp [92]; in this case, heterodimerization synergistically enhance transcription from NurRE reporters [93].

Despite the identification of these response elements and the increasing evidences that NR4A genes play a key role in different cellular functions, their downstream target genes are largely unknown. In fact, few genes other than 20{alpha}-hydroxysteroid dehydrogenase regulated by Nur77 in ovarian cells [16], and the complex regulatory functions of the three genes at different levels of hypotalamo-pituitary-adrenal axis has been characterized [94]. Recently, in the cancer cell line LNCaP, it has been shown that Nur77 can bind to a NBRE in the promoter of E2F1 increasing its transcription rate and promoting apoptosis [95], and microarray analysis shows that Nur77-mediated apoptosis in T cells involves Bcl-2-independent transcriptional activation of novel (NDG1, NDG2) and known apoptotic genes (FasL, TRAIL) [96]. However, in the vascular system, it has only been described that Nur77 regulates plasminogen activator inhibitor 1 (PAI-1) expression in ECs in response to inflammatory stimuli (TNF{alpha}, IL-1β or lipopolysaccharide) [22]. Nur77 drives transcription of PAI-1 through direct binding to a NBRE, apparently in a monomeric fashion and by a ligand-independent mechanism. Since the well-known role of PAI-1 in cardiovascular disease PAI-1 regulation by Nur77 in ECs could be a relevant link between this subfamily of genes and atherosclerosis. In fact, Nur77 colocalizes with PAI-1 in atherosclerotic lesions; however, whether it participates in the regulation of PAI-1 in other vascular cells is unknown. Inflammation seems also to trigger Nurr1 induction in synovial tissue vascular ECs, in this case mediated by corticotropin-releasing hormone (CRH), cytokines and PGE2 [51], but the target genes are unknown. Therefore, further studies are required to identify specific genes regulated by NR4A transcription factors in vascular cells.


   5. Concluding remarks
Top
 Abstract
 1. Introduction
 2. The NR4A subfamily...
 3. Regulation of NR4A...
 4. Functional role of...
 5. Concluding remarks
References
 
Factors regulating the state of differentiation and the proliferative/migratory activity of vascular cells are known to play a key role in vascular diseases such as atherosclerosis and hypertension, but the mechanisms controlling such processes are not fully understood. The novel high through-put technologies for differential gene expression analysis have allowed to expand our knowledge on the molecular basis of vascular biology. Using these techniques, genes from the NR4A subfamily, previously identified in the nervous system, have recently emerged as potentially relevant genes in the vascular wall. In different organ and tissues, these genes are involved in differentiation, proliferation and apoptosis, and they appear as novel candidates for the coordinated regulation of cell proliferation and apoptosis, essential to maintain normal architecture in different tissues among them the vascular wall. They are immediate early genes up-regulated in vascular cells by a variety of atherogenic stimuli and overexpressed in the vascular wall subjected to intravascular injury and in chronic atherosclerotic lesions. They also take part in the regulation of T cell populations and in monocyte apoptosis. These genes are induced by complex and redundant signaling pathways, but at least in vascular cells their up-regulation seems to be highly dependent on the activation state of CREB. However, contrasting functions have been attributed to NOR-1 and Nur77 as pro- and anti-proliferative, respectively, in the vascular wall. In addition, nowadays, the mechanisms linking these genes with the cell cycle machinery or the interaction with other transcription factors, which are known players in the biology of vascular cells, is poorly understood. Recent findings suggest that vascular cell gene expression is not controlled by a few cell-specific transcription factors but rather by complex interactions between multiple general and tissue-specific proteins. Therefore, the challenge for the future is not only to keep adding new genes to the list of those involved in atherogenesis, but to understand the complex relationship that these master genes establish to regulate vascular biology and cardiovascular disease.


   Acknowledgements
 
We apologize to colleagues whose work has not been directly cited due to space limitations. Original work was supported by grants from Fondo de Investigaciones Sanitarias PI020361 and Freedom to Discover Grant from BMS (USA). We thank J. Rius (Postdoctoral fellow) and X. Crespo (Predoctoral fellow) for their participation in our studies on NR4A genes.


   Notes
 
Time for primary review 29 days


   References
Top
 Abstract
 1. Introduction
 2. The NR4A subfamily...
 3. Regulation of NR4A...
 4. Functional role of...
 5. Concluding remarks
 References
 

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