Nur77 Protein Research Articles

Nur77 induced by HIF-1α mediates vascular remodeling in hypoxic pulmonary hypertension.

Nur77 is a member of the NR4A subfamily of orphan nuclear receptors that is expressed and has a function within the immune system. This study aimed to investigate the role of Nur77 in hypoxic pulmonary hypertension. SPF male SD rats were exposed in hypobaric chamber simulating 5000 m high altitude for 0, 3, 7, 14, 21 or 28 days. Rat pulmonary artery smooth muscle cells (RPASMCs) were cultured under normoxic conditions (5% CO2-95% ambient air) or hypoxic conditions (5% O2 for 6 h, 12 h, 24 h, 48 h). Hypoxic rats developed pulmonary arterial remodeling and right ventricular hypertrophy with significantly increased pulmonary arterial pressure. The levels of Nur77, HIF-1α and PNCA were upregulated in pulmonary arterial smooth muscle from hypoxic rats. Silencing of either Nur77 or HIF-1α attenuated hypoxia-induced proliferation. Silencing of HIF-1α down-regulated Nur77 protein level, but Nur77 silence did not reduce HIF-1α. Nur77 was not con-immunoprecipitated with HIF-1α. This study demonstrated that Nur77 acted as a downstream regulator of HIF-1α under hypoxia, and plays a critical role in the hypoxia-induced pulmonary vascular remodeling, which is regulated by HIF-1α. Nur77 maybe a novel target of HPH therapy.

In Silico Discovery of Stapled Peptide Inhibitor Targeting the Nur77-PPARγ Interaction and Its Anti-Breast-Cancer Efficacy.

The binding of peroxisome proliferator-activated receptor γ (PPARγ) to the orphan nuclear receptor Nur77 facilitates the ubiquitination and degradation of Nur77, and leads to aberrant fatty acid uptake for breast cancer progression. Because of its crucial role in clinical prognosis, the interaction between Nur77 and PPARγ is an attractive target for anti-breast-cancer therapy. However, developing an inhibitor of the Nur77-PPARγ interaction poses a technical challenge due to the absence of the crystal structure of PPARγ and its corresponding interactive model with Nur77. Here, ST-CY14, a stapled peptide,is identified as a potent modulator of Nur77 with a KD value of 3.247×10-8M by in silico analysis, rational design, and structural modification. ST-CY14 effectively increases Nur77 protein levels by blocking the Nur77-PPARγ interaction, thereby inhibiting lipid metabolism in breast tumor cells. Notably, ST-CY14 significantly suppresses breast cancer growth and bone metastasis in mice. The findings demonstrate the feasibility of exploiting directly Nur77-PPARγ interaction in breast cancer, and generate what to the best knowledge is the first direct inhibitor of the Nur77-PPARγ interaction available for impeding fatty acid uptake and therapeutic development.

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.

Functional Interaction Between α-Synuclein and Nurr1 in Dopaminergic Neurons

Increased expression of alpha-synuclein (ASYN) and decreased expression of Nurr1 are associated with Parkinson’s disease (PD) pathogenesis. These two proteins interact functionally and ASYN overexpression suppresses Nurr1 levels. ASYN pan-neuronal overexpression coupled with Nurr1 hemizygosity followed by Nurr1 repression in aging mice results in the manifestation of a typical PD-related phenotype and pathology. Here we investigated in mice the effects of C-terminally truncated ASYN(120) overexpression in dopaminergic (DA-ergic) neurons compounded with Nurr1 hemizygosity (‘2-hit-DA’). We report that ‘2-hit-DA’ animals did not manifest a characteristic PD-related phenotype, despite further substantia nigra ASYN-overexpression-dependent and age dependent Nurr1 protein downregulation. However, they displayed increased energy expenditure, reduced striatal dopamine (DA) and prolonged hyperactivity to a novel environment indicating impaired habituation. This DA-ergic dysfunction was observed in young adult ‘2-hit-DA’ mice, persisted throughout life and it was associated with ASYN and Nurr1 synergistic alterations of DAT levels and function. Our experiments indicate that the expression levels of ASYN and Nurr1 are critical in the dysregulation of the nigrostriatal DA system and may be involved in neuropsychiatric aspects of PD.

Nur77 promotes invasion and migration of gastric cancer cells through the NF-κB/IL-6 pathway

To analyze the association of Nur77 with overall survival of gastric cancer patients and investigate the role of Nur77 in invasion and migration of gastric cancer cells. Oncomine database was used to analyze the expression of Nur77 in gastric cancer and gastric mucosa tissues, and the distribution characteristics of Nur77 protein between gastric cancer and normal tissues were compared using Human Protein Atlas. GEPIA2 was used to analyze the relationship of Nur77 expression and the patients' survival. The expression of Nur77 in gastric cancer cell lines GES-1, AGS and MKN-45 were detected by Western blotting. The regulatory interactions between IL-6 and Nur77 were verified by transfecting the cells with specific Nur-77 siRNA and Nur-77-overexpressing plasmid. The changes in migration ability of the cells following Nur-77 knockdown were assessed with scratch assay. The effect of Nur-77 overexpression or IL-6 knockdown, or their combination, on migration and invasion of the gastric cancer cells were examined using Transwell assay. The effect of Nur77 expression level on NF-κB/IL-6 pathway activation was analyzed using Western blotting. Oncomine database showed that gastric cancer tissues expressed a significantly higher level of Nur77 mRNA than normal tissues (P < 0.05). Nur77 expression was detected mostly in the nucleus, and a high Nur77 expression was associated with a poor survival outcome of the patients (P < 0.05). In gastric cancer cells, the high expression of Nur77 participated in the regulation of IL-6. Nur77 silencing significantly lowered the migration ability of the cells (P < 0.05), and IL-6 silencing significantly attenuated the enhanced migration caused by Nur77 overexpression (P < 0.05). Nur77 participates in the activation of NF-κB/IL-6 signaling pathway by regulating the expression of p-p65, p65, p-Stat3 and Stat3. A high Nur77 expression is strongly correlated with a poor prognosis of gastric cancer patients. Nur77 promotes the invasion and migration of gastric cancer cells possibly by regulating the NF-κB/IL-6 signaling pathway.

Embelin and levodopa combination therapy for improved Parkinson's disease treatment.

Parkinson’s disease (PD), a progressive neurodegenerative disorder, affects dopaminergic neurons. Oxidative stress and gut damage play critical roles in PD pathogenesis. Inhibition of oxidative stress and gut damage can prevent neuronal death and delay PD progression. The objective of this study was to evaluate the therapeutic effect of embelin or the combination with levodopa (LD) in a rotenone-induced PD mouse model. At the end of experimentation, the mice were sacrificed and the midbrain was used to evaluate various biochemical parameters, such as nitric oxide, peroxynitrite, urea, and lipid peroxidation. In the substantia nigra (midbrain), tyrosine hydroxylase (TH) expression was examined by immunohistochemistry, and Nurr1 expression was evaluated by western blotting. Gut histopathology was evaluated on tissue sections stained with hematoxylin and eosin. In silico molecular docking studies of embelin and α-synuclein (α-syn) fibrils were also performed. Embelin alone or in combination with LD ameliorated oxidative stress and gut damage. TH and Nurr1 protein levels were also significantly restored. Docking studies confirmed the affinity of embelin toward α-syn. Taken together, embelin could be a promising drug for the treatment of PD, especially when combined with LD.

γ-Glutamylcysteine attenuates amyloid-β oligomers-induced neuroinflammation in microglia via blocking NF-κB signaling pathway

Alzheimer's disease (AD) is the most prevalent neurogenerative disease, characterized by progressive memory loss and cognitive deficits. Intracellular neurofibrillary tangles (NFTs) and amyloid-β (Aβ)-formed neuritic plaques are major pathological features of AD. Aβ evokes activation of microglia to release inflammatory mediators and ROS to induce neurotoxicity, leading to neurodegeneration. γ-Glutamylcysteine (γ-GC), an intermediate dipeptide of the GSH-synthesis pathway with anti-inflammatory and anti-oxidative properties, represents a relatively unexplored option for AD treatment. In the present study, we investigated the anti-inflammatory effect of γ-GC on Aβ oligomer (AβO)-induced neuroinflammation and the associated molecular mechanism in microglia. The results showed that γ-GC reduced AβO-induced release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and nitric oxide (NO), and the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). γ-GC decreased ROS and MDA production and increased the GSH level, GSH/GSSG ratio, and SOD activity in AβO-treated microglia. Mechanistically, γ-GC inhibited activation of nuclear factor kappa B (NF-κB), and upregulated the nuclear receptor-related 1 (Nurr1) protein expression to suppress the transcriptional effect of NF-κB on the inflammatory genes. Besides, γ-GC suppressed the AβO-induced neuroinflammation in mice. These findings suggested that γ-GC might represent a potential therapeutic agent for anti-neuroinflammation.

Regulating mitochondrial homeostasis and inhibiting inflammatory responses through Celastrol.

BackgroundThe high morbidity and mortality rate of coronary heart disease poses a serious threat to human health. Atherosclerosis, a chronic inflammation of the blood vessel wall, is a significant pathological process leading to coronary heart disease. Macrophage inflammation plays a crucial role in the occurrence and development of atherosclerosis.MethodsMacrophage inflammation model was constructed by lipopolysaccharide (LPS), and macrophages were treated with Celastrol at different concentrations (0, 0.1, 1, 10, 100 ng/mL) and different time points (0, 1, 3, 6, 12 h). Real-time quantitative PCR (qPCR) and Western Blot were used to detect the expression of Nur77 mRNA and protein. Macrophages were then pretreated with 100 nmol/L tripterine for 40min and co-cultured with 100 ng/mL LPS. The expression levels of inflammatory factors and chemokines, phosphorylation of phospho-dynamin-related protein 1 (p-Drp1) at Ser637 and expression of mitochondrial fusion protein mitochondrial fusion protein mitofusin-2 (Mfn2) were detected by qPCR, Western blot and ELISA, respectively. The changes of mitochondrial membrane potential were detected by JC-1 probe.Results100 nmol/L Celastrol can significantly inhibit LPS-induced inflammatory responses and down-regulate the expression levels of cytokines such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), tumor necrosis factor-α (TNF-α), chemokines (CCL-2, and CXCL-10), as well as chemokines. And Celastrol could regulate mitochondrial fission and fusion by promoting the phosphorylation of the Drp1 at the Ser637 site, thereby inhibiting mitochondrial fission. At the same time, by up-regulating the level of the Mfn2, Celastrol also promoted mitochondrial fusion. In addition, we found that the nuclear factor-k-gene binding (NF-κB), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 signaling pathways aided the drug’s anti-inflammatory effects. We also explored the relationship between Celastrol and the nuclear receptor Nur77 and found that it could up-regulate the expression of Nur77.ConclusionsOur study found that Celastrol could reduce inflammation by regulating Drp1 dependent mitochondrial fission and fusion, as well as the ERK1/2, p38, NF-κB signaling pathways. This finding provides a strong direction for the development of new anti-inflammatory drugs for atherosclerosis.

CB2 receptor activation inhibits the phagocytic function of microglia through activating ERK/AKT-Nurr1 signal pathways.

Neuroinflammation is closely related to the pathogenesis of neurodegenerative diseases. Activation of microglia, the resident immune cells in CNS, induces inflammatory responses, resulting in the release of neurotoxic molecules, which favors neuronal death and neurodegeneration. Nuclear receptor-related 1 (Nurr1) protein, one of the orphan nuclear receptor superfamilies, is an emerging target for neuroprotective therapy. In addition, the anti-inflammatory function of cannabinoid (CB) receptors has attracted increasing interest. As both CB receptors (especially CB2 receptor) and Nurr1 exist in microglia, and regulate a number of same molecular points suchas NF-κB, we herein explored the interplay between the CB2 receptor and Nurr1 as well as the regulatory mechanisms in microglial cells. We showed that the application of CB2 receptor agonists JWH015 (1, 10 μM) significantly increased the nuclear Nurr1 protein in BV-2 cells and primary midbrain microglia. Overexpression of Nurr1 or application of Nurr1 agonist C-DIM12 (10 μM) significantly increased the mRNA level of CB2 receptor in BV-2 cells, suggesting that positive expression feedback existing between the CB2 receptor and Nurr1. After 2-AG and JWH015 activated the CB2 receptors, the levels of p-ERK, p-AKT, p-GSK-3β in BV-2 cells were significantly increased. Using ERK1/2 inhibitor U0126 and PI3K/AKT inhibitor LY294002, we revealed that the amount of Nurr1 in the nucleus was upregulated through β-arrestin2/ERK1/2 and PI3K/AKT/GSK-3β signaling pathways. With these inhibitors, we found a cross-talk interaction between the two pathways, and the ERK1/2 signaling pathway played a more dominant regulatory role. Furthermore, we demonstrated that when the CB2 receptor was activated, the phagocytic function of BV-2 cells was significantly weakened;the activation of Nurr1 also inhibited the phagocytic function of BV-2 cells. Pretreatment with the signaling pathway inhibitors, especially U0126, reversed the inhibitory effect of 2-AG on phagocytosis, suggesting that CB2 receptor may regulate the phagocytic function of microglia by activating Nurr1. In conclusion, CB2 receptor or/and Nurr1-mediated signal pathways play instrumental roles in the progress of phagocytosis, which are expected to open up new treatment strategies for neurodegenerative diseases.

Effects of 3, 4-divanillyltetrahydrofuran from Urtica fissa on sexual dysfunction in diabetic mice

Ethnopharmacological relevanceUrtica fissa E. Pritz. are important herbs and have been traditionally used as ethnic medicine to treat rheumatism, inflammation, diabetes, and benign prostatic hyperplasia by the Han, Uighur, and other minorities in China, and also as an aphrodisiac in Uighur medicine. Aims of the studyTo determine the effect and potential mechanism of 3, 4-divanillyltetrahydrofuran (DVTF), one of the main active components isolated from U. fissa on hypogonadism in diabetic mice. Materials and methodsThe active compound DVTF was extracted and separated from the roots of U. fissa and identified using mass spectrometry and nuclear magnetic resonance spectroscopy. A mouse model of diabetes was established using high fat and sugar diet combined with streptozotocin. In the treatment groups, mice were received different doses of DVTF for 4 weeks. Fasting blood glucose levels, physiological and biochemical indices, and the mating behavior of DM mice were analyzed. Changes in testicular morphology were assessed using light microscopy and transmission electron microscopy. The expression of testosterone synthesis-related signaling proteins was detected using western blotting. Molecular docking was used to determine the binding ability of DVTF to Nur77. ResultsIn diabetic mice, body weight and fasting blood glucose levels decreased. Mating behavior, including mount latency, mount number, and intromission number, was improved following DVTF treatment. Plasma total testosterone, free testosterone, and insulin resistance were positively associated with the recovery of testicular pathological structures in diabetic mice. DVTF treatment increased the expression of Nur77, StAR, and P450scc in the testes of diabetic mice. DVTF and Nur77 formed chemical bonds at five sites. ConclusionAs one of the main active components of U. fissa, DVTF exert potential therapeutic effects on testicular injury and hypogonadism caused by diabetes through activating the expression of Nur77 and testosterone synthesis related proteins. Our result will provide new insight for the clinical application of Urtica fissa E. Pritz., especially DVTF, as a potential drug candidate in the treatment of hypogonadism in diabetes.

Variability in Behavioral Phenotypes after Forced Swimming-Induced Stress in Rats Is Associated with Expression of the Glucocorticoid Receptor, Nurr1, and IL-1β in the Hippocampus.

Individual differences in coping with stress may determine either a vulnerable or resilient phenotype. Therefore, it is important to better understand the biology underlying the behavioral phenotype. We assessed whether individual behavioral phenotype to acute stress is related with the hippocampal expression of glucocorticoid receptor (GR), Nurr1, interleukin-1 beta (IL-1β) or brain-derived neurotrophic factor (BDNF). Wistar male rats were exposed to forced swimming for 15 min and sacrificed at different times. Behavioral response was analyzed, and it was compared with the gene and protein expression of GR, Nurr1, IL-1β and BDNF in the hippocampus for each time point. Behavioral phenotyping showed a group with high immobility (vulnerable) while another had low immobility (resilient). No significant differences were found in the Nurr1, IL-1β and BDNF mRNA levels between resilient and vulnerable rats at different recovery times except for Nr3c1 (gene for GR). However, exposure to stress caused significantly higher levels of GR, Nurr1 and IL-1β proteins of vulnerable compared to resilient rats. This variability of behavioral phenotypes is associated with a differential molecular response to stress that involves GR, Nurr1, and IL-1β as mediators in coping with stress. This contributes to identifying biomarkers of susceptibility to stress.

Deletion of pleiotrophin impairs glucose tolerance and liver metabolism in pregnant mice: Moonlighting role of glycerol kinase.

Pleiotrophin is a pleiotropic cytokine that has been demonstrated to have a critical role in regulating energy metabolism, lipid turnover and plasticity of adipose tissue. Here, we hypothesize that this cytokine can be involved in regulatory processes of glucose and lipid homeostasis in the liver during pregnancy. Using 18-days pregnant Ptn-deficient mice, we evaluated the biochemical profile (circulating variables), tissue mRNA expression (qPCR) and protein levels of key enzymes and transcription factors involved in main metabolic pathways. Ptn deletion was associated with a reduction in body weight gain, hyperglycemia and glucose intolerance. Moreover, we observed an impairment in glucose synthesis and degradation during late pregnancy in Ptn-/- mice. Hepatic lipid content was significantly lower (73.6%) in Ptn-/- mice and was associated with a clear reduction in fatty acid, triacylglycerides and cholesterol synthesis. Ptn deletion was accompanying with a diabetogenic state in the mother and a decreased expression of key proteins involved in glucose and lipid uptake and metabolism. Moreover, Ptn-/- pregnant mice have a decreased expression of transcription factors, such as PPAR-α, regulating lipid uptake and glucose and lipid utilization. Furthermore, the augmented expression and nuclear translocation of glycerol kinase, and the decrease in NUR77 protein levels in the knock-out animals can further explain the alterations observed in hepatic glucose metabolism. Our results point out for the first time that pleiotrophin is an important player in maintaining hepatic metabolic homeostasis during late gestation, and further highlighted the moonlighting role of glycerol kinase in the regulation of maternal glucose homeostasis during pregnancy.

Malayoside, a cardenolide glycoside extracted from Antiaris toxicaria Lesch, induces apoptosis in human non-small lung cancer cells via MAPK-Nur77 signaling pathway

Lung cancer is the leading cause of cancer deaths in the world. Non-small cell lung cancer (NSCLC), with poor prognosis and resistance to chemoradiotherapy, is the most common histological type of lung cancer. Therefore, it is necessary to develop new and more effective treatment strategy for NSCLC. Nur77, an orphan member of the nuclear receptor superfamily, induces apoptosis in cancer cells including NSCLC cells, by high expression and translocation to mitochondria. Small molecules trigger expression and mitochondrial localization of Nur77 may be an ideal anti-cancer drug candidate. Here, we report malayoside, a cardiac glycoside in the extract of Antiaris toxicaria Lesch., had different sensitivities to NSCLC cells. Malayoside induced apoptosis in NCI-H460 cells. Meanwhile, malayoside induced Nur77 expression and mitochondrial localization, and its induction of apoptosis was Nur77-dependent. To investigate the molecular mechanism of malayoside inducing Nur77 and apoptosis, we found that malayoside activated MAPK signaling pathway, including both ERK and p38 phosphorylation. The suppression of MAPK signaling activation inhibited the expression of Nur77 and apoptosis induced by malayoside. Our studies in nude mice showed that malayside potently inhibited the growth of tumor cells in vivo. Furthermore, the anti-cancer effect of malayosidwas in vivo was also related to the elevated expression of Nur77, p-ERK, and p-p38 proteins. Our results suggest that malayoside possesses an anti-NSCLC activity in vitro and in vivo mainly via activation of MAPK-Nur77 signaling pathway, indicating that malayoside is a promising chemotherapeutic candidate for NSCLC.

Quantitative Immunohistochemistry to Measure Regional Expression of Nurr1 in the Brain and the Effect of the Nurr1 Heterozygous Genotype.

The transcription factor Nurr1 is a member of the steroid hormone nuclear receptor superfamily. Ablation of Nurr1 expression arrests mesencephalic dopamine neuron differentiation while attenuation of Nurr1 in the subiculum and hippocampus impairs learning and memory. Additionally, reduced Nurr1 expression has been reported in patients with Parkinson’s disease and Alzheimer’s disease. In order to better understand the overall function of Nurr1 in the brain, quantitative immunohistochemistry was used to measure cellular Nurr1 protein expression, across Nurr1 immunoreactive neuronal populations. Additionally, neuronal Nurr1 expression levels were compared between different brain regions in wild-type mice (+/+) and Nurr1 heterozygous mice (+/−). Regional Nurr1 protein was also investigated at various time points after a seizure induced by pentylenetetrazol (PTZ). Nurr1 protein is expressed in various regions throughout the brain, however, a wide range of Nurr1 expression levels were observed among various neuronal populations. Neurons in the parietal and temporal cortex (secondary somatosensory, insular, auditory, and temporal association cortex) had the highest relative Nurr1 expression (100%) followed closely by the claustrum/dorsal endopiriform cortex (85%) and then subiculum (76%). Lower Nurr1 protein levels were found in neurons in the substantia nigra pars compacta and ventral tegmental area (39%) followed by CA1 (25%) and CA3 (19%) of the hippocampus. Additionally, in the parietal and temporal cortex, two distinct populations of high and medium Nurr1 expressing neurons were observed. Comparisons between +/− and +/+ mice revealed Nurr1 protein was reduced in +/− mice by 27% in the parietal/temporal cortex, 49% in the claustrum/dorsal endopiriform cortex, 25% in the subiculum, 33% in substantia nigra pars compacta, 22% in ventral tegmental area, and 21% in CA1 region of the hippocampus. Based on these data, regional mechanisms appear to exist which can compensate for a loss of a Nurr1 allele. Following a single PTZ-induced seizure, Nurr1 protein in the dentate gyrus peaked around 2 h and returned to baseline by 8 h. Since altered Nurr1 expression has been implicated in neurologic disorders and Nurr1 agonists have showed protective effects, understanding regional protein expression of Nurr1, therefore, is necessary to understand how changes in Nurr1 expression can alter brain function.

The orphan nuclear receptor Nur77 plays a vital role in BPA-induced PC12 cell apoptosis

Bisphenol A (BPA) is a typical environmental endocrine disruptor that can migrate into organisms through skin contact, breathing, diet and various other approaches. The reproductive toxicity and neurotoxicity of BPA has been confirmed by several toxicological studies. However, the neurotoxicity of BPA is still controversial. In the present study, we used PC12 cells as a model to investigate the mechanism of BPA-induced neuronal apoptosis. BPA exposure reduced cell viability, altered cell morphology and aggravated intracellular Lactate dehydrogenase (LDH) release, intracellular Ca2+ concentration, Reactive oxygen species (ROS) levels, apoptosis and the reduction in the mitochondrial transmembrane potential (ΔΨm). Moreover, the results of the Western blot (WB) and Real-time quantitative polymerase chain reaction (RT-qPCR) assays indicated that the expression levels of Nur77 in the BPA group were down-regulated and accompanied by the downregulation of the NF-κb/Bcl-2 proteins and the upregulation of cleaved-caspase 3, which is a marker of apoptosis. However, these changes were significantly reversed with the upregulation of the Nur77 protein by introducing plasmids carrying the nur77 gene. These results indicated that BPA-induced apoptosis was closely related to Nur77-mediated inhibition of the NF-κb/Bcl-2 pathway.

Cantharidin Induces Apoptosis and Promotes Differentiation of AML Cells Through Nuclear Receptor Nur77-Mediated Signaling Pathway.

BackgroundAcute myeloid leukemia (AML) is a hematopoietic malignancy characterized by uncontrolled proliferation and accumulation of myeloblasts in the bone marrow (BM), blood, and other organs. The nuclear receptors Nur77 is a common feature in leukemic blasts and has emerged as a key therapeutic target for AML. Cantharidin (CTD), a main medicinal component of Mylabris (blister beetle), exerts an anticancer effect in multiple types of cancer cells.PurposeThis study aims to characterize the anti-AML activity of CTD in vitro and in vivo and explore the potential role of Nur77 signaling pathway.Study Design/MethodsThe inhibition of CTD on cell viability was performed in different AML cells, and then the inhibition of CTD on proliferation and colony formation was detected in HL-60 cells. Induction of apoptosis and promotion of differentiation by CTD were further determined. Then, the potential role of Nur77 signaling pathway was assessed. Finally, anti-AML activity was evaluated in NOD/SCID mice.ResultsIn our study, CTD exhibited potent inhibition on cell viability and colony formation ability of AML cells. Moreover, CTD significantly induced the apoptosis, which was partially reversed by Z-VAD-FMK. Meanwhile, CTD promoted the cleavage of caspases 8, 3 and PARP in HL-60 cells. Furthermore, CTD obviously suppressed the proliferation and induced the cell cycle arrest of HL-60 cells at G2/M phase. Meanwhile, CTD effectively promoted the differentiation of HL-60 cells. Notably, CTD transiently induced the expression of Nur77 protein. Interestingly, CTD promoted Nur77 translocation from the nucleus to the mitochondria and enhanced the interaction between Nur77 and Bcl-2, resulting in the exposure of the BH3 domain of Bcl-2, which is critical for the conversion of Bcl-2 from an antiapoptotic to a proapoptotic protein. Importantly, silencing of Nur77 attenuated CTD-induced apoptosis, reversed CTD-mediated cell cycle arrest and differentiation of HL-60 cells. Additionally, CTD also exhibited an antileukemic effect in NOD/SCID mice with the injection of HL-60 cells into the tail vein.ConclusionsOur studies suggest that Nur77-mediated signaling pathway may play a critical role in the induction of apoptosis and promotion of differentiation by CTD on AML cells.

Genome-Wide Analysis Identifies NURR1-Controlled Network of New Synapse Formation and Cell Cycle Arrest in Human Neural Stem Cells.

Nuclear receptor-related 1 (Nurr1) protein has been identified as an obligatory transcription factor in midbrain dopaminergic neurogenesis, but the global set of human NURR1 target genes remains unexplored. Here, we identified direct gene targets of NURR1 by analyzing genome-wide differential expression of NURR1 together with NURR1 consensus sites in three human neural stem cell (hNSC) lines. Microarray data were validated by quantitative PCR in hNSCs and mouse embryonic brains and through comparison to published human data, including genome-wide association study hits and the BioGPS gene expression atlas. Our analysis identified ~40 NURR1 direct target genes, many of them involved in essential protein modules such as synapse formation, neuronal cell migration during brain development, and cell cycle progression and DNA replication. Specifically, expression of genes related to synapse formation and neuronal cell migration correlated tightly with NURR1 expression, whereas cell cycle progression correlated negatively with it, precisely recapitulating midbrain dopaminergic development. Overall, this systematic examination of NURR1-controlled regulatory networks provides important insights into this protein’s biological functions in dopamine-based neurogenesis.

Nur77 knock-down suppresses glioma by regulating CXCR4

IntroductionThe aim of this study is to explain the effects and mechanisms of Nur77 in glioma development.Material and methodsNur77 protein and gene expression were measured by immunohistochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) in tissues from glioma and craniocerebral injury patients. The patients were divided into two groups according to median Nur77 gene expression and the correlations between clinical pathology, progression-free survival (PFS), overall survival (OS), and Nur77 gene expression were analysed. U257 and U87 cells were employed to assess the effects of Nur77 in cancer cell development by MTT, flow cytometry, and transwell and wound healing assays. Relative protein and gene expression were evaluated by Western blot assay and RT-qPCR, and PI3K nuclear volumes were evaluated by cellular immunofluorescence to explain the relevant mechanisms.ResultsCompared with that in normal tissues, Nur77 protein and gene expression in glioma tissues was significantly up-regulated (p &lt; 0.01); this up-regulation increased with increasing tumour stage (p &lt; 0.01). Compared with the high Nur77 expression group, the low Nur expression group showed significantly improved PFS and OS (both p &lt; 0.01). Nur77 gene expression appeared to be closely correlated with clinical pathology. In vitro studies of si-Nur77 transfection revealed a significant reduction in the cell viability of the si-Nur77 group, with increasing apoptosis rate (p &lt; 0.001). Nur77 knock-down resulted in significant downregulation of cell invasion and wound healing rates in the si-Nur77 group (p &lt; 0.01), with remarkable reductions in CXCR4 and PI3K.ConclusionsNur77 is an important oncogene in glioma, and Nur77 expression is closely correlated with glioma clinical pathology, PFS, and OS. Nur77 knock-down exerts inhibitory effects on glioma by regulating CXCR4/PI3K.

Striatal Nurr1, but not FosB expression links a levodopa-induced dyskinesia phenotype to genotype in Fisher 344 vs. Lewis hemiparkinsonian rats

Numerous genes, and alterations in their expression, have been identified as risk factors for developing levodopa-induced dyskinesia (LID). However, our understanding of the complexities of molecular changes remains insufficient for development of clinical treatment. In the current study we used gene array, in situ hybridization, immunohistochemistry, and microdialysis to provide a unique compare and contrast assessment of the relationship of four candidate genes to LID, employing three genetically distinct rat strains (Sprague-Dawley (SD), Fischer-344 (F344) and Lewis-RT.1) showing differences in dyskinesia susceptibility and ‘first-ever LID’ versus ‘chronic LID’ expression in subjects displaying equal dyskinesia severity. In these studies, rat strains were easily distinguishable for their LID propensity with: 1) a majority of SD rats expressing LID (LID+) and a subset being resistant (LID-); 2) all F344 rats readily developing (LID+); and 3) all Lewis rats being LID-resistant (LID-). Following chronic levodopa, LID+ SD rats showed significant increases in candidate gene expression: Nr4a2/(Nurr1) > > Trh > Inhba = Fosb. However, SD rats with long-standing striatal dopamine (DA) depletion treated with first-ever versus chronic high-dose levodopa revealed that despite identical levels of LID severity: 1) Fosb and Nurr1 transcripts but not protein were elevated with acute LID expression; 2) FOSB/ΔFOSB and NURR1 proteins were elevated only with chronic LID; and 3) Trh transcript and protein were elevated only with chronic LID. Strikingly, despite similar levodopa-induced striatal DA release in both LID-expressing F344 and LID-resistant Lewis rats, Fosb, Trh, Inhba transcripts were significantly elevated in both strains; however, Nurr1 mRNA was significantly increased only in LID+ F344 rats. These findings suggest a need to reevaluate currently accepted genotype-to-phenotype relationships in the expression of LID, specifically that of Fosb, a transcription factor generally assumed to play a causal role, and Nurr1, a transcription factor that has received significant attention in PD research linked to its critical role in the survival and function of midbrain DA neurons but who's striatal expression, generally below levels of detection, has remained largely unexplored as a regulator of LID. Finally these studies introduce a novel ‘model’ (inbred F344 vs inbred Lewis) that may provide a powerful tool for investigating the role for ‘dyskinesia-resistance’ genes downstream of ‘dyskinesia-susceptibility’ genes in modulating LID expression, a concept that has received considerably less attention and offers a new ways of thinking about antidyskinetic therapies.

Change of Nurr1 expression in mouse hippocampal CA3 region following excitotoxic neuronal damage.

Objective(s):Nuclear receptor-related protein 1 (Nurr1), one of immediate-early genes, is a member of orphan nuclear receptor family. The aim of this study was to investigate the time-dependent change of Nurr1 protein expression in the mouse hippocampal CA3 region following kainic acid (KA)-induced excitotoxic neuronal damage.Materials and Methods:Male ICR mice were used as experimental animals, and 30 mg/kg KA was administered intraperitoneally. To confirm the KA-induced neuronal damage in the hippocampal CA3 region, Fluoro-Jade B histofluorescence staining was performed. In addition, the time-dependent change of Nurr1 protein expression was also examined using immunohistochemistry and western blot analysis. Results:Marked neuronal damage was observed in the hippocampal CA3 region at 24 hr after KA injection. In addition, both Nurr1 immunoreactivity and protein level were significantly increased at 6 hr and 12 hr after KA injection, and then decreased at 24 hr after KA injection.Conclusion:This result indicates that KA-induced alteration of Nurr1 protein expression may be associated with the neuronal degeneration in the hippocampal CA3 region after KA injection.