Orphan Nuclear Receptor Nur77 Research Articles

Chloroquine modulates inflammatory autoimmune responses through Nurr1 in autoimmune diseases

For over a half-century the anti-malarial drug chloroquine (CQ) has been used as a therapeutic agent, alone or in combination, to treat autoimmune diseases. However, neither the underlying mechanism(s) of action nor their molecular target(s) are well defined. The orphan nuclear receptor Nurr1 (also known as NR4A2) is an essential transcription factor affecting the development and maintenance of midbrain dopaminergic neurons. In this study, using in vitro T cell differentiation models, we demonstrate that CQ activates TREG cell differentiation and induces Foxp3 gene expression in a Nurr1-dependent manner. Remarkably, CQ appears to induce Nurr1 function by two distinct mechanisms: firstly, by direct binding to Nurr1’s ligand-binding domain and promoting its transcriptional activity and secondly by upregulation of Nurr1 expression through the CREB signaling pathway. In contrast, CQ suppressed gene expression and differentiation of pathogenic TH17 cells. Importantly, using a valid animal model of inflammatory bowel disease (IBD), we demonstrated that CQ promotes Foxp3 expression and differentiation of TREG cells in a Nurr1-dependent manner, leading to significant improvement of IBD-related symptoms. Taken together, these data suggest that CQ ameliorates autoimmune diseases via regulating Nurr1 function/expression and that Nurr1 is a promising target for developing effective therapeutics of human inflammatory autoimmune diseases.

The orphan nuclear receptor NUR77 promotes trophoblast invasion at early pregnancy through paracrine placental growth factor.

NR4A1 (NUR77) is an orphan nuclear receptor that has been implicated in both cell survival and apoptosis. However, the role of NUR77 in trophoblast function during early placenta development has not been fully elucidated. In this study, we showed that NUR77 expression was significantly lower in the villi of the recurrent miscarriage (RM) group compared to that in the healthy controls (HCs) group. We used immunohistochemistry and found that NUR77 was highly expressed in human placental villi during early pregnancy, especially in syncytiotrophoblast (STB), and was expressed at a much lower level in STB from the RM group than in those from HC group. Western blotting data further confirmed that NUR77 was highly expressed in primary human term placental STB and the FSK-induced BeWo cell line. Moreover, antibody array screening and ELISA revealed that NUR77 promoted significant placental growth factor (PGF) expression during trophoblast fusion. Ectopic overexpression and knockdown experiments demonstrated that PGF was a novel downstream target of NUR77, and serum PGF expression correlated positively with trophoblast NUR77 mRNA levels in HCs and RM patients. Importantly, bioinformatics analysis identified two NUR77 binding sites in the PGF promoter region, and chromatin immunoprecipitation (ChIP) coupled with Western blotting analysis further verified that NUR77 bound directly to the PGF promoter region and promoted PGF expression. Furthermore, in a BeWo/HTR-8 co-culture system, FSK-induced BeWo-secreted PGF promoted HTR-8 cell migration and invasion, and an anti-PGF antibody reversed this effect. Collectively, these results indicated that NUR77 may play a key role in regulating trophoblast invasion at early pregnancy. KEY MESSAGES: NUR77 expression was significantly decreased in the syncytiotrophoblast of the recurrent miscarriage group compared to that in the healthy control group. NUR77 promoted PGF expression during trophoblast fusion. ChIP and western blotting experiments verified that NUR77 bound directly to the PGF promoter region and activated PGF expression in trophoblast. Trophoblast-derived PGF promoted HTR-8 cell migration and invasion in a cell co-culture system.

Human Trophoblast Differentiation Is Associated With Profound Gene Regulatory and Epigenetic Changes.

Defective placental implantation and vascularization with accompanying hypoxia contribute to preeclampsia (PE), a leading cause of maternal and neonatal morbidity and mortality. Genetic and epigenetic mechanisms underlying differentiation of proliferative cytotrophoblasts (CytTs) to multinucleated syncytiotrophoblast (SynT) are incompletely defined. The SynT performs key functions in nutrient and gas exchange, hormone production, and protection of the fetus from rejection by the maternal immune system. In this study, we used chromatin immunoprecipitation sequencing of midgestation human trophoblasts before CytT and after SynT differentiation in primary culture to analyze changes in binding of RNA polymerase II (Pol II) and of active and repressive histone marks during SynT differentiation. Our findings reveal that increased Pol II binding to promoters of a subset of genes during trophoblast differentiation was closely correlated with active histone marks. This gene set was enriched in those controlling immune response and immune modulation, including interferon-induced tetratricopeptide repeat and placenta-specific glycoprotein gene family members. By contrast, genes downregulated during SynT differentiation included proinflammatory transcription factors ERG1, cFOS, and cJUN, as well as members of the NR4A orphan nuclear receptor subfamily, NUR77, NURR1, and NOR1. Downregulation of proinflammatory transcription factors upon SynT differentiation was associated with decreased promoter enrichment of endogenous H3K27Ac and H3K9Ac and enhanced binding of H3K9me3 and histone deacetylase 1. However, promoter enrichment of H3K27me3 was low in both CytT and SynT and was not altered with changes in gene expression. These findings provide important insight into mechanisms underlying human trophoblast differentiation and may identify therapeutic targets for placental disorders, such as PE.

Prevention of progression of pulmonary hypertension by the Nur77 agonist 6-mercaptopurine: role of BMP signalling.

Pulmonary arterial hypertension (PAH) is a progressive fatal disease characterised by abnormal remodelling of pulmonary vessels, leading to increased vascular resistance and right ventricle failure. This abnormal vascular remodelling is associated with endothelial cell dysfunction, increased proliferation of smooth muscle cells, inflammation and impaired bone morphogenetic protein (BMP) signalling. Orphan nuclear receptor Nur77 is a key regulator of proliferation and inflammation in vascular cells, but its role in impaired BMP signalling and vascular remodelling in PAH is unknown.We hypothesised that activation of Nur77 by 6-mercaptopurine (6-MP) would improve PAH by inhibiting endothelial cell dysfunction and vascular remodelling.Nur77 expression is decreased in cultured pulmonary microvascular endothelial cells (MVECs) and lungs of PAH patients. Nur77 significantly increased BMP signalling and strongly decreased proliferation and inflammation in MVECs. In addition, conditioned medium from PAH MVECs overexpressing Nur77 inhibited the growth of healthy smooth muscle cells. Pharmacological activation of Nur77 by 6-MP markedly restored MVEC function by normalising proliferation, inflammation and BMP signalling. Finally, 6-MP prevented and reversed abnormal vascular remodelling and right ventricle hypertrophy in the Sugen/hypoxia rat model of severe angioproliferative PAH.Our data demonstrate that Nur77 is a critical modulator in PAH by inhibiting vascular remodelling and increasing BMP signalling, and activation of Nur77 could be a promising option for the treatment of PAH.

Abstract 4301: Hypoxia-induced Nur77 activates PI3K-p110a/Akt/mTOR via suppression of Dicer/let-7i-5p to induce epithelial-to-mesenchymal transition in colon cancer cells

Abstract Cancer cells reside in the hypoxic niches of tumors easily undergo epithelial-to-mesenchymal transition (EMT). We have recently shown that hypoxia-induced EMT to enhance colon cancer cell invasive growth is dependent on the orphan nuclear receptor Nur77 and is mediated by Akt signaling. However, its underlying molecular mechanisms remain elusive. Here we found that hypoxia-induced Nur77 reduced Akt phosphorylation and its downstream mediator mTOR by decreasing the mRNA stability of p110α, the catalytic subunit α of phosphatidylinositol 3-kinase. Further analyses revealed that this effect could be mediated by down-regulation of Dicer, an important microRNA (miR) processor, and decreased biogenesis of the miR, let-7i-5p. Luciferase reporter assays confirmed that let-7i-5p could directly target the 3’UTR of p110α mRNA. Knockdown of Dicer or overexpression of let-7i-5p inhibited Nur77-induced EMT phenotypes. These data uncover a novel mechanistic link connecting Nur77, Akt, and invasive properties of colon cancer in the hypoxic microenvironment. Citation Format: Sally K. Y. To, Shan Deng, Jin-Zhang Zeng, Alice Sze Tsai Wong. Hypoxia-induced Nur77 activates PI3K-p110a/Akt/mTOR via suppression of Dicer/let-7i-5p to induce epithelial-to-mesenchymal transition in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4301.

Tumor necrosis factor stimulates fibroblast growth factor 23 levels in chronic kidney disease and non-renal inflammation

Fibroblast growth factor 23 (FGF23) regulates phosphate homeostasis, and its early rise in patients with chronic kidney disease is independently associated with all-cause mortality. Since inflammation is characteristic of chronic kidney disease and associates with increased plasma FGF23 we examined whether inflammation directly stimulates FGF23. In a population-based cohort, plasma tumor necrosis factor (TNF) was the only inflammatory cytokine that independently and positively correlated with plasma FGF23. Mouse models of chronic kidney disease showed signs of renal inflammation, renal FGF23 expression and elevated systemic FGF23 levels. Renal FGF23 expression coincidedwith expression of the orphan nuclear receptor Nurr1 regulating FGF23 in other organs. Antibody-mediated neutralization of TNF normalized plasma FGF23 and suppressed ectopic renal Fgf23 expression. Conversely, TNF administration to control mice increased plasma FGF23 without altering plasma phosphate. Moreover, in Il10-deficient mice with inflammatory bowel disease and normal kidney function, plasma FGF23 was elevated and normalized upon TNF neutralization. Thus, the inflammatory cytokine TNF contributes to elevated systemic FGF23 levels and also triggers ectopic renal Fgf23 expression in animal models of chronic kidney disease.

Repeated bifenthrin exposure alters hippocampal Nurr-1/AChE and induces depression-like behavior in adult rats

Exposure to insecticides has been associated with depression-like symptoms, especially among occupationally exposed populations, such as farmers. Although the neurotoxicity of pyrethroids such as bifenthrin (BF) is well established, it is still unclear whether exposure to BF may have deleterious effects on the hippocampus and thus behavior. We verified the hypothesis that repeated exposure to BF in a rat model elicits neurochemical and behavioral alterations, the latter of which reflects depression-related symptoms. Adult male Wistar rats (n = 12 per group) were orally administered with different doses of BF (0.6 or 2.1 mg/kg body weight (b.w.)) on a daily basis for 60 days; control rats received the vehicle (corn oil). Different biochemical changes were assessed in the hippocampus, a region of the brain regulating spatial memory; behavioral tests were also conducted. Our results revealed depressive-like behaviors that were expressed by increased despair behavior in the Forced-swimming test. Repeated exposure to BF also decreased acetylcholinesterase (AChE) activity in the hippocampus and butyrylcholinesterase (BuChE) activity in plasma. A significant reduction in the activities of hippocampal membrane-bound ATPases (Na+/K+-ATPase and Mg2+-ATPase) was also observed in BF-treated rats compared to controls. Furthermore, a significant decrease in mRNA expression and protein synthesis of both AChE and orphan nuclear receptor (Nurr-1), as well as in the expression of muscarinic–cholinergic receptor (M1 mAchR) and nicotinic–cholinergic receptor (nAchR2α) was observed in the hippocampus of treated rats compared to controls. Also, BF exposure induced apoptosis as assessed by hippocampal Casp-3 protein levels. Our findings suggest that repeated exposure to BF affects hippocampal signaling and Nurr-1/AChE, and this was accompanied by depression-like state in adult rats.

Design and synthesis of biotinylated cardiac glycosides for probing Nur77 protein inducting pathway

The orphan nuclear receptor Nur77 (also known as TR3 or nerve growth factor-induced clone B NGFI-B) functions as a nuclear transcription factor in the regulation of target gene expression and plays a critical role in the regulation of differentiation, proliferation, apoptosis, and survival of many different cell types. Recent studies demonstrate that Nur77 also involves many important physiological and pathological processes including cancer, inflammation and immunity, cardiovascular diseases, and bone diseases. Our previous studies showed that cardiac glycosides could induce the expression of Nur77 protein and its translocation from the nucleus to the cytoplasm and subsequent targeting to mitochondria, leading to apoptosis of cancer cells. In order to probe the Nur77 protein inducting pathway, we designed and synthesized a series of novel biotinylated cardiac glycosides from β-Antiarin and α-Antiarin, two typical cardiac glycosides from the plant of Antiaris toxicaria. The induction of Nur77 protein expression of these biotinylated cardiac glycosides and their inhibitory effects on NIH-H460 cancer cell proliferation were evaluated. Results displayed that some biotinylated cardiac glycosides could significantly induce the expression of Nur77 protein comparable with their parent compounds β-Antiarin and α-Antiarin. Also, their streptavidin binding activities were evaluated. Among them, biotinylated cardiac glycosides P4b and P5a exhibited significant effect on the induction of Nur77 expression along with high binding capacity with streptavidin, suggesting that they can be used as probes for probing Nur77 protein inducting pathway.

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.

Epigenetic treatment-mediated modulation of PD-L1 predicts potential therapy resistance over response markers in myeloid malignancies: A molecular mechanism involving effectors of PD-L1 reverse signaling.

Recent evidence suggests an association exists between resistance to epigenetic therapy (EGT) and the expression of programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) in myeloid malignancies. Biomarkers are required to predict resistance to EGT in myeloid malignancies, which together with the delineation of associated molecular mechanisms, may provide additional understanding for novel treatment strategies when investigating resistance to EGT. The present study aimed to investigate the in vivo effects of EGT on the expression of PD-1, PD-L1 and orphan nuclear receptor NUR77 with clinical responses in patients with myeloid malignancies. In addition, in vitro and in vivo characterization of the effects of EGT on the expression of NF-κB and Bcl-xL, potential downstream targets of PD-L1 reverse signaling, were evaluated to determine components of the molecular mechanism responsible for these effects. The in vivo effects of EGT on the expression of PD-1, PD-L1 and a previously identified molecular marker of response to EGT, NUR77 was characterized in peripheral blood mononuclear cells (PBMC) from patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) treated with either azacytidine (Aza) alone or a combination of Aza and the histone deacetylase inhibitor (HDACi) LBH-589 during the first cycle of therapy. The correlation of clinical responses to treatment with EGT with the expression of PD-1, PD-L1 and NUR77 demonstrated that the induction of PD-L1 mRNA levels was associated with resistance to EGT despite the concurrent augmentation of NUR77 expression. The characterization of potential downstream effector molecules of reverse PD-L1 signaling identified EGT-mediated induction of Bcl-xL and NF-κB mRNA expression in vitro and in vivo, suggesting a potential anti-apoptotic molecular mechanism was responsible for PD-L1-mediated resistance to EGT. Taken together, these observations suggest that enhanced PD-L1 expression may confer resistance to EGT over known EGT response markers in myeloid malignancies, and provides a potential molecular mechanism involving the modulation of effectors of PD-L1 reverse signaling, which may in-part, be responsible for these effects.

Nurr1 (NR4A2) regulates Alzheimer's disease-related pathogenesis and cognitive function in the 5XFAD mouse model.

The orphan nuclear receptor Nurr1 (also known as NR4A2) is critical for the development and maintenance of midbrain dopaminergic neurons, and is associated with Parkinson's disease. However, an association between Nurr1 and Alzheimer's disease (AD)‐related pathology has not previously been reported. Here, we provide evidence that Nurr1 is expressed in a neuron‐specific manner in AD‐related brain regions; specifically, it is selectively expressed in glutamatergic neurons in the subiculum and the cortex of both normal and AD brains. Based on Nurr1’s expression patterns, we investigated potential functional roles of Nurr1 in AD pathology. Nurr1 expression was examined in the hippocampus and cortex of AD mouse model and postmortem human AD subjects. In addition, we performed both gain‐of‐function and loss‐of‐function studies of Nurr1 and its pharmacological activation in 5XFAD mice. We found that knockdown of Nurr1 significantly aggravated AD pathology while its overexpression alleviated it, including effects on Aβ accumulation, neuroinflammation, and neurodegeneration. Importantly, 5XFAD mice treated with amodiaquine, a highly selective synthetic Nurr1 agonist, showed robust reduction in typical AD features including deposition of Aβ plaques, neuronal loss, microgliosis, and impairment of adult hippocampal neurogenesis, leading to significant improvement of cognitive impairment. These in vivo and in vitro findings suggest that Nurr1 critically regulates AD‐related pathophysiology and identify Nurr1 as a novel AD therapeutic target.

Transcriptional up-regulation of relaxin-3 by Nur77 attenuates β-adrenergic agonist–induced apoptosis in cardiomyocytes

The relaxin family peptides have been shown to exert several beneficial effects on the heart, including anti-apoptosis, anti-fibrosis, and anti-hypertrophy activity. Understanding their regulation might provide new opportunities for therapeutic interventions, but the molecular mechanism(s) coordinating relaxin expression in the heart remain largely obscured. Previous work demonstrated a role for the orphan nuclear receptor Nur77 in regulating cardiomyocyte apoptosis. We therefore investigated Nur77 in the hopes of identifying novel relaxin regulators. Quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) data indicated that ectopic expression of orphan nuclear receptor Nur77 markedly increased the expression of latexin-3 (RLN3), but not relaxin-1 (RLN1), in neonatal rat ventricular cardiomyocytes (NRVMs). Furthermore, we found that the β-adrenergic agonist isoproterenol (ISO) markedly stimulated RLN3 expression, and this stimulation was significantly attenuated in Nur77 knockdown cardiomyocytes and Nur77 knockout hearts. We showed that Nur77 significantly increased RLN3 promoter activity via specific binding to the RLN3 promoter, as demonstrated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. Furthermore, we found that Nur77 overexpression potently inhibited ISO-induced cardiomyocyte apoptosis, whereas this protective effect was significantly attenuated in RLN3 knockdown cardiomyocytes, suggesting that Nur77-induced RLN3 expression is an important mediator for the suppression of cardiomyocyte apoptosis. These findings show that Nur77 regulates RLN3 expression, therefore suppressing apoptosis in the heart, and suggest that activation of Nur77 may represent a useful therapeutic strategy for inhibition of cardiac fibrosis and heart failure.

Macroporous silica nanoparticles for delivering Bcl2-function converting peptide to treat multidrug resistant-cancer cells

The abundance of B cell lymphoma gene 2 (Bcl-2) is closely correlated with the resistance of cancer cells to chemotherapeutic agents, and a peptide derived from orphan nuclear receptor Nur77 can convert Bcl-2 from a protector to a killer of cancer cells. However, successful application of the Bcl-2-converting peptide to treat drug-resistant cancer cells depends on an efficient delivery carrier. Mesoporous silica nanoparticles (MSNs) have been extensively studied as promising candidates for small molecule drug delivery. However, the effective encapsulation and intracellular delivery of peptides using small pore-sized MSNs still remain a great technical challenge. In this paper, an effective delivery platform for Bcl-2-converting peptide was fabricated by us to treat multidrug resistant-cancer cells via tuning the surface functionality of macroporous silica nanoparticles. The resulting large-sized pore silica nanoparticles, especially those modified with thiol group, exhibited the high Bcl-2-converting peptide-loading efficiency of over 40%. Moreover, the peptide induced MCF7/DOX cells into apoptotic status by penetrating cytomembrane into mitochondria and being bound with Bcl-2 to expose the BH3 domain with the aid of various surface functionalities-decorated MSNs. In particular, amine-modified surface of MSNs caused the greater influence on the cell apoptosis-inducing effects of peptide in comparison with other functionalities-modified ones. Taken together, our study, for the first time, demonstrates a special approach towards pore size and surface functionality-collectively modulated silica-based nanostructural material for effective delivery of bio-macromolecules (e.g., Bcl-2-converting peptide) to treat the multidrug resistant-cancer cells with elevated Bcl-2 levels.

The Nurr1 Ligand,1,1-bis(3'-Indolyl)-1-(p-Chlorophenyl)Methane, Modulates Glial Reactivity and Is Neuroprotective in MPTP-Induced Parkinsonism.

The orphan nuclear receptor Nurr1 (also called nuclear receptor-4A2) regulates inflammatory gene expression in glial cells, as well as genes associated with homeostatic and trophic function in dopaminergic neurons. Despite these known functions of Nurr1, an endogenous ligand has not been discovered. We postulated that the activation of Nurr1 would suppress the activation of glia and thereby protect against loss of dopamine (DA) neurons after subacute lesioning with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our previous studies have shown that a synthetic Nurr1 ligand, 1,1-bis(3′-indolyl)-1-(p-chlorophenyl)methane (C-DIM12), suppresses inflammatory gene expression in primary astrocytes and induces a dopaminergic phenotype in neurons. Pharmacokinetic analysis of C-DIM12 in mice by liquid chromatography-mass spectrometry demonstrated that approximately three times more compound concentrated in the brain than in plasma. Mice treated with four doses of MPTP + probenecid over 14 days were monitored for neurobehavioral function, loss of dopaminergic neurons, and glial activation. C-DIM12 protected against the loss of DA neurons in the substantia nigra pars compacta and DA terminals in the striatum, maintained a ramified phenotype in microglia, and suppressed activation of astrocytes. In vitro reporter assays demonstrated that C-DIM12 was an effective activator of Nurr1 transcription in neuronal cell lines. Computational modeling of C-DIM12 binding to the three-dimensional structure of human Nurr1 identified a high-affinity binding interaction with Nurr1 at the coactivator domain. Taken together, these data suggest that C-DIM12 is an activator of Nurr1 that suppresses glial activation and neuronal loss in vivo after treatment with MPTP, and that this receptor could be an efficacious target for disease modification in individuals with Parkinson’s disease and related disorders.

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.

B cell lymphoma 2 (Bcl-2) residues essential for Bcl-2's apoptosis-inducing interaction with Nur77/Nor-1 orphan steroid receptors

Apoptosis is mediated through the extrinsic or intrinsic pathway. Key regulators of the intrinsic apoptotic pathway are the family of B cell lymphoma 2 (Bcl-2) proteins. The activity of the prototypical Bcl-2 protein is usually considered antiapoptotic. However, under some conditions, Bcl-2 associates with the orphan nuclear hormone receptors Nur77 and Nor-1, converting Bcl-2 into a proapoptotic molecule. Expression of Nur77 and Nor-1 is induced by a variety of signals, including those leading to apoptosis. Translocation of Nur77/Nor-1 to mitochondria results in their association with Bcl-2, exposing the Bcl-2 homology (BH) 3 domain and causing apoptosis. However, the molecular details of this interaction are incompletely understood. Here, through extensive Bcl-2 mutagenesis and functional assays, we identified residues within Bcl-2 that are essential for its interaction with Nur77/Nor-1. Although an initial report has suggested that an unstructured loop region between the Bcl-2 BH4 and BH3 domains is required for Bcl-2's interaction with Nur77/Nor-1, we found that it is dispensable for this interaction. Instead, we found important interacting residues at the BH4 domain and crucial interacting residues between the BH1 and BH2 domains. Bcl-2 alanine mutants at this region could no longer interact with Nur77/Nor-1 and could not initiate Nur77/Bcl-2-mediated cell death. However, they still retained their anti-apoptotic capability in two different death assays. These results establish crucial residues in Bcl-2 required for Nur77/Nor-1-mediated apoptosis and point to potential new strategies for manipulating Bcl-2 function.

Theca Cell INSL3 and Steroids Together Orchestrate the Growing Bovine Antral Follicle.

Insulin-like peptide 3 (INSL3) and its specific receptor RXFP2 are both expressed by theca interna cells of the growing antral follicle where they form an essential regulatory element in the production of the steroid precursor androstenedione. Using primary cultures of bovine theca cells from the mid follicular phase together with steroid agonists and antagonists we have examined how ovarian steroids modulate INSL3 expression. Transcript analysis shows that these cells express estrogen receptors α and β, androgen and progesterone receptors, besides the orphan nuclear receptors SF1 and nur77. Whereas, exogenous androgens have little or no effect, the androgen antagonist bicalutamide stimulates INSL3 production. In contrast, estrogen receptor agonists, as also progesterone, are stimulatory. Importantly, estrogen receptor signaling is convergent with the protein kinase A signaling pathway activated by LH, such that the estrogen receptor antagonist can inhibit the mild stimulatory effect of LH, and vice versa the PKA antagonist H89 blocks stimulation by estradiol. A significant finding is that the major steroid metabolite androstenedione appears to act predominantly as an estrogen and not an androgen in this system. Transfection of INSL3 gene promoter-reporter constructs together with various steroid receptor expression plasmids supports these findings and shows that steroid action uses non-classical pathways not requiring canonical steroid-responsive elements in the proximal promoter region. Together, the results indicate that increasing estrogens in the follicular phase stimulate a feedforward loop driving INSL3 signaling and thereby promoting steroidogenesis in the growing antral follicle until the LH surge which effectively switches off INSL3 expression.

Nur77 Is Upregulated in B-1a Cells by Chronic Self-Antigen Stimulation and Limits Generation of Natural IgM Plasma Cells.

B-1a cells are a unique population of innate-like B cells with a highly restricted and self-reactive BCR repertoire. Preimmune "natural" IgM produced by B-1a-derived plasma cells is essential for homeostatic clearance of cellular debris and forms a primary layer of protection against infection. In this study, we take advantage of a fluorescent reporter of BCR signaling to show that expression of the orphan nuclear hormone receptor Nur77 is upregulated under steady-state conditions in self-reactive B-1a cells in response to chronic Ag stimulation. Nur77-deficient mice exhibit elevated natural serum IgM (but not IgG) and marked expansion of IgM plasma cells of B-1a origin. Moreover, we show that Nur77 restrains the turnover of B-1a cells and the accumulation of immature IgM plasma cells. Thus, we identify a new critical negative-regulatory pathway that serves to maintain B-1a cells in a quiescent state in the face of chronic endogenous Ag stimulation.

Ginsenoside 20(S)-Rh2 Induces Apoptosis and Differentiation of Acute Myeloid Leukemia Cells: Role of Orphan Nuclear Receptor Nur77.

Ginsenoside 20(S)-Rh2 has been shown to induce apoptosis and differentiation of acute myeloid leukemia (AML) cells. However, the underlying molecular mechanisms are not fully understood. In our study, 20(S)-Rh2 induced the expression of orphan nuclear receptor Nur77 and death receptor proteins Fas, FasL, DR5, and TRAIL, as well as the cleavage of caspase 8 and caspase 3 in HL-60 cells. Importantly, shNur77 attenuated 20(S)-Rh2-induced apoptosis and Fas and DR5 expression. Meanwhile, 20(S)-Rh2 promoted Nur77 translocation from the nucleus to mitochondria and enhanced the interaction between Nur77 and Bcl-2, resulting in the exposure of the BH3 domain of Bcl-2 and activation of Bax. Furthermore, 20(S)-Rh2 promoted the differentiation of HL-60 cells as evidenced by Wright-Giemsa staining, NBT reduction assay, and detection of the myeloid differentiation marker CD11b by flow cytometry. Notably, shNur77 reversed 20(S)-Rh2-mediated HL-60 differentiation. Additionally, 20(S)-Rh2 also exhibited an antileukemic effect and induced Nur77 expression in NOD/SCID mice with the injection of HL-60 cells into the tail vein. Together, our studies suggest that the Nur77-mediated signaling pathway is highly involved in 20(S)-Rh2-induced apoptosis and differentiation of AML cells.

Modulation of the Nur77-Bcl-2 apoptotic pathway by p38α MAPK.

Orphan nuclear receptor Nur77 promotes apoptosis by targeting mitochondria through interaction with Bcl-2, an event that converts Bcl-2 from a survival to killer. However, how the Nur77-Bcl-2 apoptotic pathway is regulated remains largely unknown. In this study, we examined the regulation of the Nur77-Bcl-2 pathway by CCE9, a xanthone compound. Our results demonstrated that the apoptotic effect of CCE9 depended on its induction of Nur77 expression, cytoplasmic localization, and mitochondrial targeting. The activation of the Nur77-Bcl-2 pathway by CCE9 was associated with its activation of p38α MAPK. Inhibition of p38α MAPK activation by knocking down or knocking out p38α MAPK impaired the effect of CCE9 on inducing apoptosis and the expression and cytoplasmic localization of Nur77. In addition, CCE9 activation of p38α MAPK resulted in Bcl-2 phosphorylation and Bcl-2 interaction with Nur77, whereas inhibition of p38α MAPK activation or expression suppressed the interaction. Moreover, mutating Ser87 and Thr56 in the loop of Bcl-2, which are known to be phosphorylated by p38α MAPK, impaired the ability Bcl-2 to interact with Nur77. Together, our results reveal a profound role of p38α MAPK in regulating the Nur77-Bcl-2 apoptotic pathway through its modulation of Nur77 expression, Bcl-2 phosphorylation, and their interaction.