Orphan Receptor Nur77 Research Articles

The orphan receptor Nur77 binds cytoplasmic LPS to activate the non-canonical NLRP3 inflammasome

Intracellular sensing of lipopolysaccharide (LPS) by murine caspase-11 or human caspase-4 initiates a protease cascade, termed the non-canonical inflammasome, that results in gasdermin D (GSDMD) processing and subsequent NLRP3 inflammasome activation. In an effort aimed at identifying additional sensors for intracellular LPS by biochemical screening, we identified the nuclear orphan receptor Nur77 as an LPS-binding protein in macrophage lysates. Nr4a1-/- macrophages exhibited impaired activation of the NLRP3 inflammasome, but not caspase-11, in response to LPS. Biochemical mapping revealed that Nur77 bound LPS directly through a domain in its C terminus. Yeast two-hybrid assays identified NLRP3 as a binding partner for Nur77. The association between Nur77 and NLRP3 required the presence of LPS and dsDNA. The source of dsDNA was the mitochondria, requiring the formation of gasdermin-D pores. Invivo, Nur77 deficiency ameliorated host response to endotoxins. Thus, Nur77 functions as an intracellular LPS sensor, binding mitochondrial DNA and LPS to activate the non-canonical NLRP3 inflammasome.

Engineered celastrol and plasmid co-delivery for in situ expression and targeted mitochondrial relocation of Nur77 protein towards effective drug resistance reversion

Overexpression of Bcl-2 (B-cell lymphoma-2) is one of the important reasons for the drug resistance of tumor cells, which often leads to the failure of clinical cancer treatment. In recent years, studies have shown that the nuclear orphan receptor Nur77 can interact with Bcl-2 to change the Bcl-2 protein from an anti-apoptotic conformation to a pro-apoptotic conformation, thereby inducing apoptosis. However, since Nur77 needs to migrate from the nucleus to the mitochondria to reverse the anti-apoptotic effect of Bcl-2, it is difficult to effectively activate the Nur77-Bcl-2 apoptotic pathway by the existing system. Herein, we design an intelligent drug delivery system (PPP/Nur77/Cela) in which drug molecular-guided gene expression products actively act on Bcl-2 proteins, which consists of a mPEG-PCL-PEI (methoxy poly(ethylene glycol)-poly(ε-caprolactone)-poly(ethylene imine), or PPP) polymer loaded with Nur77 gene and celastrol (Cela) via electrostatic and hydrophobic interactions. The system can effectively improve solubility and toxicity of Cela, and can express Nur77 efficiently in cells with a gene transfection efficiency comparable to that of PEI. Interestingly, besides being an anticancer drug, Cela can also promote Nur77 to transfer to mitochondria and convert Bcl-2 from anti-apoptotic protein to pro-apoptotic protein, thereby increasing the sensitivity of Bcl-2 overexpression cancer cells to drugs, which achieves efficient cancer treatment through complementary synergistic chemotherapy and gene therapy. To the best of our knowledge, this is a pioneering strategy to use drug molecules to guide gene expression products to actively act on target proteins, which is a truly successful realization of complementary intensive chemotherapy and gene therapy in the treatment of drug-resistant tumors.

Retraction Note: The nuclear orphan receptor Nur77 alleviates palmitate-induced fat accumulation by down-regulating G0S2 in HepG2 cells

Retraction Note: The nuclear orphan receptor Nur77 alleviates palmitate-induced fat accumulation by down-regulating G0S2 in HepG2 cells

Transcriptomics study on mechanism of Aconiti Lateralis Radix Praeparata in treatment of rats with acute heart failure

In this study,transcriptomics technique was used to investigate the mechanism of action of Aconiti Lateralis Radix Praeparata on acute heart failure rats induced by propafenone hydrochloride.First,rats were randomly divided into normal group,model group and administration group(1.25,2.5,5 g·kg-1).A rat with acute heart failure was constructed by intravenous femoral administration of proparone hydrochloride.The changes of heart rate,+dp/dtmaxand-dp/dtmaxat 5,10,20,30 and 60 min were recorded.Then another group of rats were given the same drug delivery method.In another group of animals,serum TNF-α could be determined by ELISA with the same dosage method.High-throughput sequencing technology was used to detect all gene expression differences in cardiac tissue samples of rats with acute heart failure.Through functional annotation and enrichment analysis,gene expression signaling pathways of rats with acute heart failure and rats with post-administration heart failure were screened out.The results showed that heart rate and LV+dp/dtmaxand LV-dp/dtmaxwere significantly decreased in the model group(P<0.05),while heart rate and LV+dp/dtmax and LV-dp/dtmaxwere significantly increased in the drug group(P<0.05,P<0.01).Moreover,ANP,BNP and TNF-α in acute heart failure rats was significantly decreased in high-dose aconite decoction group(P<0.05).Transcriptomics analysis showed that the mechanism of action was mainly related to activation of PI3 K-AKT signaling pathway and Jak-STAT pathway.Compared with the model group,aconite decoction up-regulated the expression of phosphatidylinostol 3-kinase(PI3 K),lysophosphatidic acid(LAP3),Bcl-3 and STAT genes,and down-regulated the expression of integrin(ITGA),nuclear orphan receptor(Nur77) genes.It could be concluded that the mechanism of aconite in treating acute heart failure rats may be related to the regulation of the PI3 k-Akt/Jak-STAT pathway.

RETRACTED ARTICLE: The Nuclear Orphan Receptor Nur77 Alleviates Palmitate-induced Fat Accumulation by Down-regulating G0S2 in HepG2 Cells

Excessive triglyceride accumulation in hepatocytes is the hallmark of obesity-associated nonalcoholic fatty liver disease (NAFLD). Elevated levels of the saturated free fatty acid palmitate in obesity are a major contributor to excessive hepatic lipid accumulation. The nuclear orphan receptor Nur77 is a transcriptional regulator and a lipotoxicity sensor. Using human HepG2 hepatoma cells, this study aimed to investigate the functional role of Nur77 in palmitate-induced hepatic steatosis. The results revealed that palmitate significantly induced lipid accumulation and suppressed lipolysis in hepatocytes. In addition, palmitate significantly suppressed Nur77 expression and stimulated the expression of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes. Nur77 overexpression significantly reduced palmitate-induced expression of PPARγ and its target genes. Moreover, Nur77 overexpression attenuated lipid accumulation and augmented lipolysis in palmitate-treated hepatocytes. Importantly, G0S2 knockdown significantly attenuated lipid accumulation and augmented lipolysis in palmitate-treated hepatocytes, whereas G0S2 knockdown had no effect on the palmitate-induced expression of Nur77, PPARγ, or PPARγ target genes. In summary, palmitate suppresses Nur77 expression in HepG2 cells, and Nur77 overexpression alleviates palmitate-induced hepatic fat accumulation by down-regulating G0S2. These results display a novel molecular mechanism linking Nur77-regulated G0S2 expression to palmitate-induced hepatic steatosis.

Synthesis and Biological Activity of Octaketides from the Cytosporone Family

AbstractCytosporone B and its simple analogues (e. g., amoitone B) were recently identified as unique agonists for nuclear orphan receptor Nur77. In this study, we have developed an aldehyde C‐H activation method for preparing natural and designer analogues from the cytosporone family. In addition, a comprehensive investigation of the compounds’ biological activities and pharmacological liabilities has been conducted and new structure activity relationships (SAR) were derived from the data. Most notably, we found that increased cytotoxicity can be obtained by virtue of fluorine substitution on the hydrophobic acyl chain.

In vivo and in silico studies to identify mechanisms associated with Nurr1 modulation following early life exposure to permethrin in rats

The present work was designed to study the mechanisms associated with Nurr1 modulation following early life permethrin (PERM) treatment during rat’s life span. Here we demonstrate that PERM exposure in rats, at a dose close to No Observed Adverse Effect Level (NOAEL) for 15days during neonatal brain development leads to its accumulation long after exposure. In striatum from adolescent rats we detected an increase in DNA methyltransferases (DNMTs) such as DNMT1, DNMT3a, Tyrosine hydroxylase, monomeric and aggregated α-synuclein protein levels. Adult rats showed enhanced DNMT3b and α-synuclein aggregation compared to the control group, while with aging a significant decrease in all biomarkers studied was observed. No changes in Nurr1 promoter methylation in adolescent, adult and old rats were found.In silico studies showed clear evidence of a strong binding interaction between PERM and its metabolite 3-phenoxybenzoic acid with the nuclear orphan receptor Nurr1. These findings suggest that an additional interference with the dopaminergic neuron pathway could occur in situ during PERM accumulation in brain.Therefore, Nurr1 modulation in early life PERM-treated rats, depends on age-related adaptive responses in animals.

Nur77 attenuates endothelin-1 expression via downregulation of NF-κB and p38 MAPK in A549 cells and in an ARDS rat model.

Acute respiratory distress syndrome (ARDS) is characterized by inflammatory injury to the alveolar and capillary barriers that results in impaired gas exchange and severe acute respiratory failure. Nuclear orphan receptor Nur77 has emerged as a regulator of gene expression in inflammation, and its role in the pathogenesis of ARDS is not clear. The objective of this study is to investigate the potential role of Nur77 and its underlying mechanism in the regulation of endothelin-1 (ET-1) expression in lipopolysaccharide (LPS)-induced A549 cells and an ARDS rat model. We demonstrate that LPS induced Nur77 expression and nuclear export in A549 cells. Overexpression of Nur77 markedly decreased basal and LPS-induced ET-1 expression in A549 cells, whereas knockdown of Nur77 increased the ET-1 expression. LPS-induced phosphorylation and nuclear translocation of NF-κB and p38 MAPK were blocked by Nur77 overexpression and augmented by Nur77 knockdown in A549 cells. In vivo, LPS induced Nur77 expression in lung in ARDS rats. Pharmacological activation of Nur77 by cytosporone B (CsnB) inhibited ET-1 expression in ARDS rats, decreased LPS-induced phosphorylation of NF-κB and p38 MAPK, and relieved lung, liver, and kidney injury. Pharmacological deactivation of Nur77 by 1,1-bis-(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH, C-DIM8) had no effect on ET-1 expression and lung injury. These results indicated that Nur77 decreases ET-1 expression by suppressing NF-κB and p38 MAPK in LPS-stimulated A549 cells in vitro, and, in an LPS-induced ARDS rat model, CsnB reduced ET-1 expression and lung injury in ARDS rats.

Molecular characterization and analysis of the porcine NURR1 gene

Orphan receptor NURR1 (also termed NR4A2) belongs to the nuclear receptor superfamily and functions as a regulatory factor of differentiation, migration, maturation and maintenance of mesencephalic dopaminergic neurons. NURR1 plays an important role in nigrostriatal dopamine neuron development and is therefore implicated in the pathogenesis of neurodegenerative diseases linked to the dopamine system of the midbrain.Here we report the isolation and characterization of porcine NURR1 cDNA. The NURR1 cDNA was RT-PCR cloned using NURR1-specific oligonucleotide primers derived from in silico sequences. The porcine NURR1 cDNA encodes a polypeptide of 598 amino acids, displaying a very high similarity with bovine, human and mouse (99%) NURR1 protein. Expression analysis revealed a differential NURR1 mRNA expression in various organs and tissues. NURR1 transcripts could be detected as early as at 60 days of embryo development in different brain tissues. A significant increase in NURR1 transcript in the cerebellum and a decrease in NURR1 transcript in the basal ganglia was observed during embryo development. The porcine NURR1 gene was mapped to chromosome 15. Two missense mutations were found in exon 3, the first coding exon of NURR1. Methylation analysis of the porcine NURR1 gene body revealed a high methylation degree in brain tissue, whereas methylation of the promoter was very low. A decrease in DNA methylation in a discrete region of the NURR1 promoter was observed in pig frontal cortex during pig embryo development. This observation correlated with an increase in NURR1 transcripts. Therefore, methylation might be a determinant of NURR1 expression at certain time points in embryo development.

Nur77-deficiency in bone marrow-derived macrophages modulates inflammatory responses, extracellular matrix homeostasis, phagocytosis and tolerance.

BackgroundThe nuclear orphan receptor Nur77 (NR4A1, TR3, or NGFI-B) has been shown to modulate the inflammatory response of macrophages. To further elucidate the role of Nur77 in macrophage physiology, we compared the transcriptome of bone marrow-derived macrophages (BMM) from wild-type (WT) and Nur77-knockout (KO) mice.ResultsIn line with previous observations, SDF-1α (CXCL12) was among the most upregulated genes in Nur77-deficient BMM and we demonstrated that Nur77 binds directly to the SDF-1α promoter, resulting in inhibition of SDF-1α expression. The cytokine receptor CX3CR1 was strongly downregulated in Nur77-KO BMM, implying involvement of Nur77 in macrophage tolerance. Ingenuity pathway analyses (IPA) to identify canonical pathways regulation and gene set enrichment analyses (GSEA) revealed a potential role for Nur77 in extracellular matrix homeostasis. Nur77-deficiency increased the collagen content of macrophage extracellular matrix through enhanced expression of several collagen subtypes and diminished matrix metalloproteinase (MMP)-9 activity. IPA upstream regulator analyses discerned the small GTPase Rac1 as a novel regulator of Nur77-mediated gene expression. We identified an inhibitory feedback loop with increased Rac1 activity in Nur77-KO BMM, which may explain the augmented phagocytic activity of these cells. Finally, we predict multiple chronic inflammatory diseases to be influenced by macrophage Nur77 expression. GSEA and IPA associated Nur77 to osteoarthritis, chronic obstructive pulmonary disease, rheumatoid arthritis, psoriasis, and allergic airway inflammatory diseases.ConclusionsAltogether these data identify Nur77 as a modulator of macrophage function and an interesting target to treat chronic inflammatory disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2469-9) contains supplementary material, which is available to authorized users.

Coalition of Nuclear Receptors in the Nervous System

A universal signaling module has been described which utilizes the nuclear form of Fibroblast growth Factor Receptor 1 (FGFR1) in a central role directing the post-mitotic development of neural cells through coordinated gene expression. In this review, we discuss in detail the current knowledge of FGFR1 nuclear interaction partners in three scenarios: (i) Engagement of FGFR1 in neuronal stem cells and regulation of neuronal differentiation; (ii) interaction with the orphan receptor Nurr1 in development of mesencephalic dopaminergic neurons; (iii) modulation of nuclear FGFR1 interactions downstream of nerve growth factor (NGF) signaling. These coalitions demonstrate the versatility of non-canonical, nuclear tyrosine kinase signaling in diverse cellular differentiation programs of neurons.

Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson’s disease

Parkinson's disease (PD), primarily caused by selective degeneration of midbrain dopamine (mDA) neurons, is the most prevalent movement disorder, affecting 1-2% of the global population over the age of 65. Currently available pharmacological treatments are largely symptomatic and lose their efficacy over time with accompanying severe side effects such as dyskinesia. Thus, there is an unmet clinical need to develop mechanism-based and/or disease-modifying treatments. Based on the unique dual role of the nuclear orphan receptor Nurr1 for development and maintenance of mDA neurons and their protection from inflammation-induced death, we hypothesize that Nurr1 can be a molecular target for neuroprotective therapeutic development for PD. Here we show successful identification of Nurr1 agonists sharing an identical chemical scaffold, 4-amino-7-chloroquinoline, suggesting a critical structure-activity relationship. In particular, we found that two antimalarial drugs, amodiaquine and chloroquine stimulate the transcriptional function of Nurr1 through physical interaction with its ligand binding domain (LBD). Remarkably, these compounds were able to enhance the contrasting dual functions of Nurr1 by further increasing transcriptional activation of mDA-specific genes and further enhancing transrepression of neurotoxic proinflammatory gene expression in microglia. Importantly, these compounds significantly improved behavioral deficits in 6-hydroxydopamine lesioned rat model of PD without any detectable signs of dyskinesia-like behavior. These findings offer proof of principle that small molecules targeting the Nurr1 LBD can be used as a mechanism-based and neuroprotective strategy for PD.

The role of the Bcl-2 BH3 domain in thymocyte apoptosis (P4416)

Abstract Over-expression of Bcl-2 has been shown to protect thymocytes from apoptosis by many stimuli except strong T cell receptor signals during negative selection. This might be due to Bcl-2 taking on a pro-apoptotic function during negative selection mediated through its BH3 domain, which is exposed upon interaction with the nuclear orphan receptor Nur77. To test this, we generated a T cell-specific transgenic mouse over-expressing Bcl-2 with a mutated BH3 domain. Consistent with our hypothesis, we observed that thymocytes expressing the Bcl-2 BH3 transgene were better protected from apoptosis by TCR stimuli in vitro than those expressing a wild-type transgene. To evaluate TCR-induced apoptosis in vivo, the Bcl-2 BH3 mutant transgenic mice were crossed to F5 and HY TCR transgenic mice. Vibratome cut thymic slices were treated acutely with F5 and HY TCR-specific peptides, circumventing the peripheral cytotoxic effects of in vivo peptide injection. Bcl-2 BH3 mutant transgenic thymocytes were almost completely rescued from peptide-induced apoptosis, but surprisingly, thymocytes expressing the wild-type Bcl-2 transgene were equivalently protected. Thus our results indicate that Bcl-2 has some pro-apoptotic function, but in contrast to previously published data in TCR-transgenic models, we conclude that Bcl-2 over-expression affords substantial protection from apoptosis accompanying negative selection.

Nuclear Import and Export Signals Control the Subcellular Localization of Nurr1 Protein in Response to Oxidative Stress*

Orphan receptor Nurr1 participates in the acquisition and maintenance of the dopaminergic cell phenotype, modulation of inflammation, and cytoprotection, but little is known about its regulation. In this study, we report that Nurr1 contains a bipartite nuclear localization signal (NLS) within its DNA binding domain and two leucine-rich nuclear export signals (NES) in its ligand binding domain. Together, these signals regulate Nurr1 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nurr1 is mostly nuclear. A Nurr1 mutant lacking the NLS failed to enter the nucleus. The Nurr1 NLS sequence, when fused to green fluorescent protein, led to nuclear accumulation of this chimeric protein, indicating that this sequence was sufficient to direct nuclear localization of Nurr1. Furthermore, two NES were characterized in the ligand binding domain, whose deletion caused Nurr1 to accumulate predominantly in the nucleus. The Nurr1 NES was sensitive to CRM1 and could function as an independent export signal when fused to green fluorescent protein. Sodium arsenite, an agent that induces oxidative stress, promoted nuclear export of ectopically expressed Nurr1 in HEK293T cells, and the antioxidant N-acetylcysteine rescued from this effect. Similarly, in dopaminergic MN9D cells, arsenite induced the export of endogenous Nurr1, resulting in the loss of expression of Nurr1-dependent genes. This study illustrates that Nurr1 shuttling between the cytosol and nucleus is controlled by specific nuclear import and export signals and that oxidative stress can unbalance the distribution of Nurr1 to favor its cytosolic accumulation.

The Nuclear Orphan Receptor Nur77 Is a Lipotoxicity Sensor Regulating Glucose-Induced Insulin Secretion in Pancreatic β-Cells

The NR4A orphan nuclear receptors Nur77, Nurr1, and Nor1 exert multiple cellular and metabolic functions. These transcriptional regulators are activated in response to extracellular stresses, including lipotoxic fatty acids (FA) and proinflammatory cytokines. The contribution of NR4As to β-cell pathophysiology is, however, unknown. We have therefore examined the role of NR4As as downstream contributors to FA-induced β-cell dysfunctions. Human pancreatic islets and insulinoma β-cells were used to determine transcriptional programs elicited by NR4A, which were compared to those triggered by palmitate treatment. Functional studies evaluated the consequence of an increased NR4A expression on insulin biosynthesis and secretion and cell viability in insulinoma β-cells. FA and cytokine treatment increased NR4A expression in pancreatic β-cells, with Nur77 being most highly inducible in murine β-cells. Nur77, Nurr1, or Nor1 modulated common and distinct clusters of genes involved notably in cation homeostasis and insulin gene transcription. By altering zinc homeostasis, insulin gene transcription, and secretion, Nur77 was found to be a major transcriptional mediator of part of FA-induced β-cell dysfunctions. The repressive role of Nur77 in insulin gene regulation was tracked down to protein-protein interaction with FoxO1, a pivotal integrator of the insulin gene regulatory network. The present study identifies a member of the NR4A nuclear receptor subclass, Nur77/NR4A1, as a modulator of pancreatic β-cell biology. Together with its previously documented role in liver and muscle, its role in β-cells establishes Nur77 as an important integrator of glucose metabolism.

Abstract 2296: Identification of novel nuclear receptor targets in estrogen receptor negative breast cancer

Abstract Introduction: Estrogen receptor (ER)-negative breast cancer has a particularly poor prognosis, and there are limited targeted agents for this subtype. Aims and Methods: To identify nuclear receptor (NR) targets, we did microarray analysis of 227 ER-negative tumors as assessed by immunohistochemistry, and performed hierarchical clustering using 41 NRs, and prediction analysis of microarrays (PAM) across the clustered groups. Cell lines were matched to identified groups using previously published data. Clinical correlations were made using previously published breast cancer datasets. Results and Conclusion: The 41 NRs clustered the tumors into 5 distinct groups. We found that the androgen receptor (AR) was the highest ranked discriminator of these groups followed by ERα. Group 1 (14% of the tumors) was characterized by elevated ERRα and RXR expression. Group 2 (9%) was characterized by higher expression of the orphan receptor NURR1. Group 3 was the largest group (37%), and showed high level expression of PPARα. Group 4 (16%) expressed higher levels of PPARγ. Group 5 (24%) demonstrated particularly high relative expression of AR, and also showed higher expression of ERα and ERRα. We used previously published microarray data to match cell lines to the NR groups using the scoring matrix developed in the PAM analysis. These cell lines are currently being tested for response to NR agonists and antagonists in growth (MTT, soft-agar, mammosphere) and apoptotic assays. We used a previously published microarray data set with long-term follow up information to identify potential differences in outcomes for the different NR subtypes. While we found no significant differences between the groups, the sample size was quite low for each subtype (n∼15 per group). In summary, we have identified several novel ER-negative tumor groups by different expression level of NRs which may contribute to the prediction of breast cancer molecular subtypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2296. doi:10.1158/1538-7445.AM2011-2296

Overexpression of the orphan receptor Nur77 and its translocation induced by PCH4 may inhibit malignant glioma cell growth and induce cell apoptosis

In previous study, n-butylidenephthalide (BP), a natural compound from Angelica sinensis, has anti-glioblastoma multiform (GBM) cell effects. In this study, we modified BP structure to increase anti-GBM cell effects. The anti-GBM cell effects of one derivative of BP, (Z)-N-(2-(dimethylamino)ethyl)-2-(3-((3-oxoisobenzofuran-1(3H)-ylidene)methyl)phenoxy)acetamide (PCH4) were tested in vitro and in vivo. MTT assay and PI/Annexin V assay were performed to evaluate the anti-GBM effects of PCH4. The Nur77 expression and translocation were assayed by RT-PCR and Western blot. The Nur77 siRNA was used to downregulate the Nur77 expression. The JNK inhibitor (SP600125) was used to block the JNK pathway. The anti-GBM effect of PCH4 is four times more than BP. The IC(50) of PCH4 on DBTRG-05MG cells was 50 µg/ml. Nur77 expression and translocation from the nucleus to the cytoplasm were important in PCH4-induced apoptosis. Furthermore, the downregulation of PCH4-induced Nur77 expression by Nur77 siRNA reduced PCH4-induced apoptosis. In addition, PCH4-induced apoptosis was associated with the JNK pathway. The JNK inhibitor, SP600125, inhibited Nur77 mRNA expression and reduced PCH4-induced apoptosis. In conclusion, PCH4, a derivative of BP, induced Nur77-mediated apoptosis via the JNK pathway and this mechanism, which is different from that of BP, may explain the increase in the anti-tumor effects on GBM.

Nur77 and Nurr1, Two Novel Players In MSC Migration and Immune Function.

AbstractAbstract 3854Mesenchymal stromal cells (MSC) have a promising potential for cellular therapies, based on their immunomodulatory functions and their differentiation capacity. However, it remains unclear how MSC exert their functions and especially homing towards effector sites is poorly understood. An imperative for future therapies is to understand the process of MSC migration. We have previously shown that only 10–20% of culture-expanded MSC is capable to migrate in vitro, but these migratory cells can not be discriminated by markers. To identify genes specific for migratory MSC, we performed a gene expression study. Using a Transwell system (12mm pore size), migrating and non-migrating fetal bone marrow MSC (FBMSC) were physically separated and RNA was extracted. MSC that were exposed to a SDF-1 gradient (0.4mm pore size) and cultured FBMSC were included as controls. Only 9 genes were differentially expressed between migratory and non-migratory MSC. This set did not include cell surface markers that enable selection for a migratory subset. The nuclear orphan receptor family members Nurr1 and Nur77 were 2-fold upregulated in migratory MSC. These results were confirmed by RQ-PCR. Nurr1 and Nur77 are members of the NR4A nuclear orphan receptor family and were first described as early response transcription factors upon growth factor stimulation. We observed that the chemokine SDF-1 and the growth factor PDGF-BB increased the expression of Nur77 and Nurr1 in MSC. To investigate the role of Nur77 and Nurr1 in migration, FBMSC were transduced with lentivirus to achieve overexpression. Both overexpression of Nur77 and Nurr1 increased specific migration towards SDF-1 compared to Mock transduced FBMSC (n=4) (Nur77 mean 297% ± 186 relative to Mock, p≤ 0.014; Nurr1 mean 227% ± 79, p≤ 0.014). We previously described that cell cycle influences MSC migration, with S- and G2/M-phase of the cell cycle negatively influencing migration. In MSC cultures overexpressing Nur77 or Nurr1, the percentage of cells in S-phase of the cell cyle was significantly reduced (Nur77 15.2±7.8%, p≤0.007; Nurr1 16.4±9.1%, p≤0.039) compared to Mock-transduced cells (24.0±8.9%). Possibly, part of the Nur77 and Nurr1 effect is regulated by an effect on cell cycle. Together these data suggest that Nur77 and Nurr1 are involved in migration of MSC, which offers perspectives to modulate migration of MSC. Because these nuclear orphan receptors were not implied in MSC biology to date, we study their immunomodulatory effect. NR4A genes modulate immune responses in a variety of cells by influencing cytokine production. The immunomodulatory effect of MSC also seems to be based on secretion of a wide variety of cytokines, chemokines and growth factors. We therefore investigated whether Nur77 and Nurr1 play a role in cytokine and growth factor production in MSC. Compared to Mock transduced MSC, overexpression of Nur77 and Nurr1 in MSC resulted in increased basal levels of IL-6 protein (Nur77 3.4±2.4 fold, p≤0.007; Nurr1 3.8±3.1 ns) and IL-8 protein (Nur77 5.9±3.3 fold, p≤0.046; Nurr1 4.7±2.2, p≤0.031) and HGF mRNA expression (Nur77 5.4±0.8 fold, p≤0.021; Nurr1 3.4±1.18, p≤0.026). This was further enhanced upon stimulation with TNFα for 24hrs relative to the unstimulated Mock (IL-6 Mock 22.9±19.5 fold; Nur77 35.1±21.9 fold; Nurr1 35.8±24.1 fold; IL-8 Mock 58±37 fold; Nur77 160±141 fold; Nurr1 262±181 fold). HGF mRNA levels were slightly elevated by stimulation with IFNγ for 24hrs (Mock 1.8±1.6 fold; Nur77 7.0±3.8 fold; Nurr1 7.1±1.7 fold; ns). Expression levels of IDO1 and TGF-ß, also involved in immune modulation by MSC, were not influenced by Nur77 or Nurr1. In conclusion, we identified two novel genes involved in MSC migration. Intriguingly these genes are also involved in cytokine production, suggesting that the fraction with the best migratory capacity is able to modulate the immune response at its effector site distinct from the bulk of culture expanded MSC that lack migratory capacity. Modulation of Nur77 and Nurr1 expression in MSC could therefore offer perspectives to ameliorate cellular therapy. Disclosures:No relevant conflicts of interest to declare.

Ccytosporone-B Induces NR4A1 Meditated Apoptosis In Aggressive Lymphoma Cells

AbstractAbstract 467NR4A1 (also called Nur77, TR3, or NGFI-B) and NR4A3 (Nor-1 or Minor) are two of three members of the orphan nuclear hormone receptor (NR) family referred to as Nur77 family. For both NRs no physiological ligand has been identified. These orphan NRs have been implicated in cell cycle regulation, apoptosis, inflammation, and more recently in carcinogenesis. Their function as critical tumour suppressors genes is demonstrated by the observation that the NR4A1 and NR4A3 double knock out mouse develops rapidly acute myeloid leukemia involving decreased expression of the AP-1 transcription factors JunB and c-Jun and defective extrinsic apoptotic (Fas-L and TRAIL) signalling and their reduced expression in leukemic blasts from human AML patients.NR4A1 and NR4A3 expression analyses in lymphoid malignancies revealed a down regulation of both NRs in all lymphoma cell lines analysed (Ly 1, 3, 4, 7, 8, Karpas 422 and Sc-1), in two of eight “low grade lymphoma” entities (B-CLL and FL II) and in three of five “high grade lymphoma” entities (DLBCL, FL III, MBL) and in ALLs compared to normal controls. Further expression analyses of genes involved in extrinsic (Trail and Fas-L) and intrinsic (Bim and Puma) apoptotic pathways resulted in a uniform regulation (significant positive correlation) of all four apoptotic genes to NR4A1 and NR4A3 expression suggesting that NR4A1 and NR4A3 act as putative tumour suppressor in a substantial number of lymphoma entities and that their abrogation leads to a defective extrinsic and intrinsic apoptotic signalling (1).To investigate whether the reduction of NR4A1 and NR4A3 expression is caused by epigenetic (hypermethylation of CpG islands) or genetic (mutations) aberrations, the regulatory region and coding sequence of both NRs were subjected to a comprehensive methylational and mutational analyses in primary aggressive lymphoma samples by methylation specific PCR and direct sequencing. Whereas no hypermethylation of CpG islands in the regulatory region was detected, a total of four mutations were found in four of 17 aggressive lymphoma samples (1 of 5 FLIII, 2 of 6 DLBCL-GCB and 1 of 6 DLBCL-NGCB). One mutation in the group of the DLBCL-GCB led to an amino acid exchange.To further delineate the function of NR4A1 in aggressive lymphomas, we treated three lymphoma cell lines (Karpas422, SC-1 and Ly8) with Cytosporone B (Csn-B), a natural occurring ligand of NR4A1 known to induce NR4A1 expression (2). Treatment with Csn-B led to an induction of NR4A1 expression accompanied by a marked inhibition of lymphoma cell growth in a dose dependent manner in all three lymphoma cell lines. To investigate whether Csn-B induced growth inhibition is mediated by apoptosis we performed various apoptosis assays, and cell cycle analyses in SC-1 and Ly8 cell lines. After 6h 1.5 ×10−4 M Csn-B treatment cleaved caspase 3 was detected in 91% of Sc-1 cells and in 77% of Ly8 cells as compared to 3% and 23% in untreated controls. Similarly, in the Annexin V assay Csn-B displayed a robust pro-apoptotic activity in both cell lines within 10 h (52 % in SC-1 and 67 % in Ly8 vs 2 % and 14 %) of treatment. By flow cytometry a significant higher sub-G1 DNA content (26 % in SC-1 cells and 45% in Ly8 vs 2% and 9%) could be detected. Induction of apoptosis was accompanied by a significant rise in NR4A1 expression.Our data suggest that NR4A1 is down regulated in a substantial fraction of aggressive lymphomas and that Csn-B induces NR4A1 mediated apoptosis in lymphoma cells. Hence, regulation of NR4A1 by Csn-B is a promising new target for the development of new drugs for lymphoma therapy.1. Deutsch AJA, Beham-Schmid C, Linkesch W, Neumeister P. The Nuclear Orphan Receptors NR4A1 and NR4A3 as Putative Tumour Suppressor Genes in Aggressive Non-Hodgkin's Lymphomas. ASH Annual Meeting Abstracts. 2009 November 20, 2009;114(22):2967.2. Zhan Y, Du X, Chen H, Liu J, Zhao B, Huang D, et al. Cytosporone B is an agonist for nuclear orphan receptor Nur77. Nat Chem Biol. 2008 Sep;4(9):548-56. Disclosures:No relevant conflicts of interest to declare.

Regulation of vascular smooth muscle cell proliferation by nuclear orphan receptor Nur77

It has been reported that Nur77 over-expresses in arteriosclerotic lesions and has both pro- and anti-proliferative effects on vascular smooth muscle cells (VSMCs). We investigated the physiological function of Nur77 on proliferation in VSMCs and the effects of atorvastatin on the expression of Nur77. Platelet-derived growth factor (PDGF), a key growth factor mediating VSMC proliferation in atherogenesis and post-angioplasty restenosis, was employed to induce the transcriptional regulation of Nur77 expression in VSMCs and rat carotid artery post-angioplasty restenosis models were used to investigate the effect of atorvastatin on the expression of Nur77 by immunohistochemistry, RT-PCR, and western blot methods. In cell models, we found that PDGF-B induced Nur77 mRNA expression and protein expression through ERK-MAPK-dependent signaling pathways, and atorvastatin attenuated the expression of Nur77 induced by PDGF-B. In the rat model, our data showed Nur77 was up-regulated in neointima, but down-regulated by atorvastatin. Our results indicate that Nur77 promotes VSMC proliferation, and down-regulation of Nur77 by atorvastatin suggests a novel therapy strategy for atherogenesis based on suppression of VSMC proliferation.