Claudia M. van Tiel

Nuclear Receptors Nur77, Nurr1, and NOR-1 Expressed in Atherosclerotic Lesion Macrophages Reduce Lipid Loading and Inflammatory Responses

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 α. Lentiviral overexpression of Nur77, Nurr1, or NOR-1 reduces expression and production of interleukin (IL)-1β and IL-6 proinflammatory cytokines and IL-8, macrophage inflammatory protein-1α and -1β 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.

Activation of Nuclear Receptor Nur77 by 6-Mercaptopurine Protects Against Neointima Formation

Background— Restenosis is a common complication after percutaneous coronary interventions and is characterized by excessive proliferation of vascular smooth muscle cells (SMCs). We have shown that the nuclear receptor Nur77 protects against SMC-rich lesion formation, and it has been demonstrated that 6-mercaptopurine (6-MP) enhances Nur77 activity. We hypothesized that 6-MP inhibits neointima formation through activation of Nur77. Methods and Results— It is demonstrated that 6-MP increases Nur77 activity in cultured SMCs, which results in reduced [3H]thymidine incorporation, whereas Nur77 small interfering RNA knockdown partially restores DNA synthesis. Furthermore, we studied the effect of 6-MP in a murine model of cuff-induced neointima formation. Nur77 mRNA is upregulated in cuffed arteries, with optimal expression after 6 hours and elevated expression up to 7 days after vascular injury. Local perivascular delivery of 6-MP with a drug-eluting cuff significantly inhibits neointima formation in wild-type mice. Locally applied 6-MP does not affect inflammatory responses or apoptosis but inhibits expression of proliferating cell nuclear antigen and enhances protein levels of the cell-cycle inhibitor p27Kip1 in the vessel wall. An even stronger inhibition of neointima formation in response to local 6-MP delivery was observed in transgenic mice that overexpressed Nur77. In contrast, 6-MP does not alter lesion formation in transgenic mice that overexpress a dominant-negative variant of Nur77 in arterial SMCs, which provides evidence for the involvement of Nur77-like factors. Conclusions— Enhancement of the activity of Nur77 by 6-MP protects against excessive SMC proliferation and SMC-rich neointima formation. We propose that activation of the nuclear receptor Nur77 is a rational approach to treating in-stent restenosis.

NR4A nuclear receptors are orphans but not lonesome

The NR4A subfamily of nuclear receptors consists of three mammalian members: Nur77, Nurr1, and NOR-1. The NR4A receptors are involved in essential physiological processes such as adaptive and innate immune cell differentiation, metabolism and brain function. They act as transcription factors that directly modulate gene expression, but can also form trans-repressive complexes with other transcription factors. In contrast to steroid hormone nuclear receptors such as the estrogen receptor or the glucocorticoid receptor, no ligands have been described for the NR4A receptors. This lack of known ligands might be explained by the structure of the ligand-binding domain of NR4A receptors, which shows an active conformation and a ligand-binding pocket that is filled with bulky amino acid side-chains. Other mechanisms, such as transcriptional control, post-translational modifications and protein-protein interactions therefore seem to be more important in regulating the activity of the NR4A receptors. For Nur77, over 80 interacting proteins (the interactome) have been identified so far, and roughly half of these interactions has been studied in more detail. Although the NR4As show some overlap in interacting proteins, less information is available on the interactome of Nurr1 and NOR-1. Therefore, the present review will describe the current knowledge on the interactomes of all three NR4A nuclear receptors with emphasis on Nur77.

Nuclear receptor Nurr1 is expressed in and is associated with human restenosis and inhibits vascular lesion formation in mice involving inhibition of smooth muscle cell proliferation and inflammation.

Background— Restenosis is the major drawback of percutaneous coronary interventions involving excessive activation and proliferation of vascular smooth muscle cells (SMCs). The nuclear receptor Nurr1 is an early response gene known mainly for its critical role in the development of dopamine neurons. In the present study, we investigated Nurr1 in human and experimental vascular restenosis. Methods and Results— In a prospective cohort of 601 patients undergoing percutaneous coronary intervention, including stent placement, we found a strong association between Nurr1 haplotypes and in-stent restenosis risk. Furthermore, Nurr1 is specifically expressed in human in-stent restenosis and induced in cultured human SMCs in response to serum or tumor necrosis factor-&agr;. Lentivirus-mediated gain- and loss-of-function experiments in SMCs demonstrated that overexpression of Nurr1 inhibited proliferation, consistent with increased expression of the key cell-cycle inhibitor p27Kip1, whereas Nurr1 silencing enhanced SMC growth. The tumor necrosis factor-&agr;–induced proinflammatory response of SMCs is inhibited by Nurr1, as reflected by reduced interleukin-1&bgr;, tumor necrosis factor-&agr;, and monocyte chemoattractant protein-1 expression. Consistent with our in vitro data, endogenous Nurr1 reduced wire injury–induced proliferation and vascular lesion formation in carotid arteries of ApoE−/− mice. Conclusion— Nurr1 haplotypes are associated with human restenosis risk, and Nurr1 is expressed in human in-stent restenosis. In SMCs, Nurr1 inhibits proliferation and inflammatory responses, which explains the inhibition of SMC-rich lesion formation in mice. The recently identified small-molecule drugs that enhance the activity of Nurr1 reveal this nuclear receptor as an attractive novel target for (local) intervention in restenosis.

NR4All in the vessel wall.

A number of nuclear receptors are involved in maintenance of normal vessel wall physiology as well as in pathophysiological processes such as atherosclerosis, restenosis and remodelling. Recent studies revealed a previously unrecognized function of the NR4A subfamily of nuclear receptors as key regulatory proteins in vascular disease. The NR4A subfamily comprises the members Nur77, Nurr1 and NOR-1 and in the current review a comprehensive overview is given of the data supporting functional involvement of these nuclear receptors in three major cell types in vascular (patho)physiology; endothelial cells, smooth muscle cells and monocytes-macrophages.A number of nuclear receptors are involved in maintenance of normal vessel wall physiology as well as in pathophysiological processes such as atherosclerosis, restenosis and remodelling. Recent studies revealed a previously unrecognized function of the NR4A subfamily of nuclear receptors as key regulatory proteins in vascular disease. The NR4A subfamily comprises the members Nur77, Nurr1 and NOR-1 and in the current review a comprehensive overview is given of the data supporting functional involvement of these nuclear receptors in three major cell types in vascular (patho)physiology; endothelial cells, smooth muscle cells and monocytes-macrophages.

Discovery of Small Molecule CD40–TRAF6 Inhibitors

The CD154-CD40 receptor complex plays a pivotal role in several inflammatory pathways. Attempts to inhibit the formation of this complex have resulted in systemic side effects. Downstream inhibition of the CD40 signaling pathway therefore seems a better way to ameliorate inflammatory disease. To relay a signal, the CD40 receptor recruits adapter proteins called tumor necrosis factor receptor-associated factors (TRAFs). CD40-TRAF6 interactions are known to play an essential role in several inflammatory diseases. We used in silico, in vitro, and in vivo experiments to identify and characterize compounds that block CD40-TRAF6 interactions. We present in detail our drug docking and optimization pipeline and show how we used it to find lead compounds that reduce inflammation in models of peritonitis and sepsis. These compounds appear to be good leads for drug development, given the observed absence of side effects and their demonstrated efficacy for peritonitis and sepsis in mouse models.

p27kip1–838C>A Single Nucleotide Polymorphism Is Associated With Restenosis Risk After Coronary Stenting and Modulates p27kip1 Promoter Activity

Background— The cyclin-dependent kinase inhibitor p27kip1 is a key regulator of smooth muscle cell and leukocyte proliferation in vascular disease, including in-stent restenosis. We therefore hypothesized that common genetic variations or single nucleotide polymorphisms in p27kip1 may serve as a useful tool in risk stratification for in-stent restenosis. Methods and Results— Three single nucleotide polymorphisms concerning the p27kip1 gene (−838C>A, rs36228499; −79C>T, rs34330; +326G>T, rs2066827) were determined in a cohort of 715 patients undergoing coronary angioplasty and stent placement. We discovered that the p27kip1-838C>A single nucleotide polymorphism is associated with clinical in-stent restenosis; the −838AA genotype decreases the risk of target vessel revascularization (hazard ratio, 0.28; 95% confidence interval, 0.10 to 0.77). This finding was replicated in another cohort study of 2309 patients (hazard ratio, 0.61; 95% confidence interval, 0.40 to 0.93). No association was detected between this end point and the p27kip1-79C>T and +326G>T single nucleotide polymorphisms. We subsequently studied the functional importance of the −838C>A single nucleotide polymorphism and detected a 20-fold increased basal p27kip1 transcriptional activity of the −838A allele containing promoter. Conclusions— Patients with the p27kip1-838AA genotype have a decreased risk of in-stent restenosis corresponding with enhanced promoter activity of the −838A allele of this cell-cycle inhibitor, which may explain decreased smooth muscle cell proliferation.

Deficiency of Nuclear Receptor Nur77 Aggravates Mouse Experimental Colitis by Increased NFκB Activity in Macrophages

Nuclear receptor Nur77, also referred to as NR4A1 or TR3, plays an important role in innate and adaptive immunity. Nur77 is crucial in regulating the T helper 1/regulatory T-cell balance, is expressed in macrophages and drives M2 macrophage polarization. In this study we aimed to define the function of Nur77 in inflammatory bowel disease. In wild-type and Nur77-/- mice, colitis development was studied in dextran sodium sulphate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced models. To understand the underlying mechanism, Nur77 was overexpressed in macrophages and gut epithelial cells. Nur77 protein is expressed in colon tissues from Crohn’s disease and Ulcerative colitis patients and colons from colitic mice in inflammatory cells and epithelium. In both mouse colitis models inflammation was increased in Nur77-/- mice. A higher neutrophil influx and enhanced IL-6, MCP-1 and KC production was observed in Nur77-deficient colons after DSS-treatment. TNBS-induced influx of T-cells and inflammatory monocytes into the colon was higher in Nur77-/- mice, along with increased expression of MCP-1, TNFα and IL-6, and decreased Foxp3 RNA expression, compared to wild-type mice. Overexpression of Nur77 in lipopolysaccharide activated RAW macrophages resulted in up-regulated IL-10 and downregulated TNFα, MIF-1 and MCP-1 mRNA expression through NFκB repression. Nur77 also strongly decreased expression of MCP-1, CXCL1, IL-8, MIP-1α and TNFα in gut epithelial Caco-2 cells. Nur77 overexpression suppresses the inflammatory status of both macrophages and gut epithelial cells and together with the in vivo mouse data this supports that Nur77 has a protective function in experimental colitis. These findings may have implications for development of novel targeted treatment strategies regarding inflammatory bowel disease and other inflammatory diseases.

Dual function of Pin1 in NR4A nuclear receptor activation: Enhanced activity of NR4As and increased Nur77 protein stability

Nur77, Nurr1 and NOR-1 form the NR4A subfamily of the nuclear receptor superfamily and have been shown to regulate various biological processes among which are cell survival and differentiation, apoptosis, inflammation and metabolism. These nuclear receptors have been proposed to act in a ligand-independent manner and we aim to gain insight in the regulation of NR4A activity. A yeast two-hybrid screen identified the peptidyl-prolyl isomerase Pin1 as a novel binding partner of NR4As, which was confirmed by co-immunoprecipitation. Pin1 enhances the transcriptional activity of all three NR4A nuclear receptors and increases protein stability of Nur77 through inhibition of its ubiquitination. Enhanced transcriptional activity of NR4As requires the WW-domain of Pin1 that interacts with the N-terminal transactivation domain and the DNA-binding domain of Nur77. Most remarkably, this enhanced activity is independent of Pin1 isomerase activity. A systematic mutation analysis of all 17 Ser/Thr-Pro-motifs in Nur77 revealed that Pin1 enhances protein stability of Nur77 in an isomerase-dependent manner by acting on phosphorylated Nur77 involving protein kinase CK2-mediated phosphorylation of the Ser(152)-Pro(153) motif in Nur77. Given the role of Nur77 in vascular disease and metabolism, this novel regulation mechanism provides perspectives to manipulate Nur77 activity to attenuate these processes.

The LIM-Only Protein FHL2 Reduces Vascular Lesion Formation Involving Inhibition of Proliferation and Migration of Smooth Muscle Cells

The LIM-only protein FHL2, also known as DRAL or SLIM3, has a function in fine-tuning multiple physiological processes. FHL2 is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells and conflicting data have been reported on the regulatory function of FHL2 in SMC phenotype transition. At present the function of FHL2 in SMCs in vascular injury is unknown. Therefore, we studied the role of FHL2 in SMC-rich lesion formation. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice showed accelerated lesion formation with enhanced Ki67 expression compared with wild-type (WT)-mice. Consistent with these findings, cultured SMCs from FHL2-KO mice showed increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. Overexpression of FHL2 in SMCs inhibited CyclinD1 expression and CyclinD1-knockdown blocked the enhanced proliferation of FHL2-KO SMCs. We also observed increased CyclinD1 promoter activity in FHL2-KO SMCs, which was reduced upon ERK1/2 inhibition. Furthermore, FHL2-KO SMCs showed enhanced migration compared with WT SMCs. In conclusion, FHL2 deficiency in mice results in exacerbated SMC-rich lesion formation involving increased proliferation and migration of SMCs via enhanced activation of the ERK1/2-CyclinD1 signaling pathway.