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)-1␤ and IL-6 proinflammatory cytokines and IL-8, macrophage inflammatory protein-1␣ and -1␤ 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. A therosclerosis 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. Conclusion-NR4A-factorsThe NR4A nuclear hormone receptors were first described as early response transcription factors expressed on stimulation by growth factors. 8 -...

BackgroundDisrupting the costimulatory CD40-CD40L dyad reduces atherosclerosis, but can result in immune suppression. The authors recently identified small molecule inhibitors that block the interaction between CD40 and tumor necrosis factor receptor-associated factor (TRAF) 6 (TRAF-STOPs), while leaving CD40-TRAF2/3/5 interactions intact, thereby preserving CD40-mediated immunity.ObjectivesThis study evaluates the potential of TRAF-STOP treatment in atherosclerosis.MethodsThe effects of TRAF-STOPs on atherosclerosis were investigated in apolipoprotein E deficient (Apoe−/−) mice. Recombinant high-density lipoprotein (rHDL) nanoparticles were used to target TRAF-STOPs to macrophages.ResultsTRAF-STOP treatment of young Apoe−/− mice reduced atherosclerosis by reducing CD40 and integrin expression in classical monocytes, thereby hampering monocyte recruitment. When Apoe−/− mice with established atherosclerosis were treated with TRAF-STOPs, plaque progression was halted, and plaques contained an increase in collagen, developed small necrotic cores, and contained only a few immune cells. TRAF-STOP treatment did not impair “classical” immune pathways of CD40, including T-cell proliferation and costimulation, Ig isotype switching, or germinal center formation, but reduced CD40 and β2-integrin expression in inflammatory monocytes. In vitro testing and transcriptional profiling showed that TRAF-STOPs are effective in reducing macrophage migration and activation, which could be attributed to reduced phosphorylation of signaling intermediates of the canonical NF-κB pathway. To target TRAF-STOPs specifically to macrophages, TRAF-STOP 6877002 was incorporated into rHDL nanoparticles. Six weeks of rHDL-6877002 treatment attenuated the initiation of atherosclerosis in Apoe−/− mice.ConclusionsTRAF-STOPs can overcome the current limitations of long-term CD40 inhibition in atherosclerosis and have the potential to become a future therapeutic for atherosclerosis.

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.

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 [ 3 H]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 p27 Kip1 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. (Circulation. 2007;115:493-500.)

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.

Background: Nur77 is an orphan nuclear receptor involved in vascular disease, of which the regulation of activity is poorly understood. Results: FHL2 binds Nur77 and represses its transcriptional activity by inhibition of Nur77 association with DNA. Conclusion: FHL2 acts as a co-repressor of Nur77. Significance: Our data suggest that interaction of FHL2 with Nur77 plays a pivotal role in vascular disease.

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.

The charge isomers of bovine brain PI-TP␣ (i.e. PI-TP␣I containing a phosphatidylinositol (PI) molecule and PI-TP␣II containing a phosphatidylcholine (PC) molecule) were phosphorylated in vitro by rat brain protein kinase C (PKC) at different rates. From the doublereciprocal plot, it was estimated that the V max values for PI-TP␣I and II were 2.0 and 6.0 nmol/min, respectively; the K m values for both charge isomers were about equal, i.e. 0.7 M. Phosphorylation of charge isomers of recombinant mouse PI-TP␣ confirmed that the PC-containing isomer was the better substrate. Phosphoamino acid analysis of in vitro and in vivo 32 P-labeled PI-TP␣s showed that serine was the major site of phosphorylation. Degradation of 32 P-labeled PI-TP␣ by cyanogen bromide followed by high pressure liquid chromatography and sequence analysis yielded one 32 P-labeled peptide (amino acids 104 -190). This peptide contained Ser-148, Ser-152, and the consensus PKC phosphorylation site Ser-166. Replacement of Ser-166 with an alanine residue confirmed that indeed this residue was the site of phosphorylation. This mutation completely abolished PI and PC transfer activity. This was also observed when Ser-166 was replaced with Asp, implying that this is a key amino acid residue in regulating the function of PI-TP␣. Stimulation of NIH3T3 fibroblasts by phorbol ester or platelet-derived growth factor induced the rapid relocalization of PI-TP␣ to perinuclear Golgi structures concomitant with a 2-3-fold increase in lysophosphatidylinositol levels. This relocalization was also observed for Myc-tagged wtPI-TP␣ expressed in NIH3T3 cells. In contrast, the distribution of Myc-tagged PI-TP␣(S166A) and Myc-tagged PI-TP␣(S166D) were not affected by phorbol ester, suggesting that phosphorylation of Ser-166 was a prerequisite for the relocalization to the Golgi. A model is proposed in which the PKC-dependent phosphorylation of PI-TP␣ is linked to the degradation of PI.