Early use of dexamethasone increases Nr4a1 in Kupffer cells ameliorating acute liver failure in mice in a glucocorticoid receptor-dependent manner.

BACKGROUND AND OBJECTIVE: Acute liver failure (ALF) is a type of disease with high mortality and rapid progression with no specific treatment methods currently available. Glucocorticoids exert beneficial clinical effects on therapy for ALF. However, the mechanism of this effect remains unclear and when to use glucocorticoids in patients with ALF is difficult to determine. The purpose of this study was to investigate the specific immunological mechanism of dexamethasone (Dex) on treatment of ALF induced by lipopolysaccharide (LPS)/D-galactosamine (D-GaIN) in mice. METHODS: Male C57BL/6 mice were given LPS and D-GaIN by intraperitoneal injection to establish an animal model of ALF. Dex was administrated to these mice and its therapeutic effect was observed. Hematoxylin and eosin (H&E) staining was used to determine liver pathology. Multicolor flow cytometry, cytometric bead array (CBA) method, and next-generation sequencing were performed to detect changes of messenger RNA (mRNA) in immune cells, cytokines, and Kupffer cells, respectively. RESULTS: A mouse model of ALF can be constructed successfully using LPS/D-GaIN, which causes a cytokine storm in early disease progression. Innate immune cells change markedly with progression of liver failure. Earlier use of Dex, at 0 h rather than 1 h, could significantly improve the progression of ALF induced by LPS/D-GaIN in mice. Numbers of innate immune cells, especially Kupffer cells and neutrophils, increased significantly in the Dex-treated group. In vivo experiments indicated that the therapeutic effect of Dex is exerted mainly via the glucocorticoid receptor (Gr). Sequencing of Kupffer cells revealed that Dex could increase mRNA transcription level of nuclear receptor subfamily 4 group A member 1 (Nr4a1), and that this effect disappeared after Gr inhibition. CONCLUSIONS: In LPS/D-GaIN-induced ALF mice, early administration of Dex improved ALF by increasing the numbers of innate immune cells, especially Kupffer cells and neutrophils. Gr-dependent Nr4a1 upregulation in Kupffer cells may be an important ALF effect regulated by Dex in this process.

Cytoplasmic location of NR4A1 in aggressive lymphomas is associated with a favourable cancer specific survival.

The nuclear orphan receptor NR4A1 functions as tumour suppressor in aggressive lymphomas by pro-apoptotic genomic and non-genomic effects. Here, we immunohistochemically studied the clinico-pathological relevance of NR4A1 protein expression patterns in a cohort of 60 diffuse large B cell lymphoma (DLBCL) patients and non-neoplastic lymph nodes. We observed a significant association between high cytoplasmic NR4A1 and favourable cancer-specific survival and the germinal centre B cell-like subtype, respectively. Moreover, the percentage of lymphoma cells exhibiting cytoplasmic NR4A1 significantly correlated to those showing cleaved caspase 3. Complementary, functional profiling using gene set enrichment of Reactome pathways based on publicly available microarray data was applied to determine pathways potentially implicated in cytoplasmic localization of NR4A1 and validated by means of semi quantitative real-time PCR. The pathway analysis revealed changes in the ERK1/2 pathway, and this was corroborated by the finding that high cytoplasmic NR4A1 was associated with higher expression of ERK1/2 targets in our cohort. These data indicate that high cytoplasmic NR4A1 is associated with a favourable lymphoma-specific survival and highlights the importance of NR4A1 expression patterns as potential prognostic marker for risk assessment in aggressive lymphomas.

Secreted IgM deficiency leads to increased BCR signaling that results in abnormal splenic B cell development.

Mice lacking secreted IgM (sIgM -/-) antibodies display abnormal splenic B cell development, which results in increased marginal zone and decreased follicular B cell numbers. However, the mechanism by which sIgM exhibit this effect is unknown. Here, we demonstrate that B cells in sIgM -/- mice display increased B cell receptor (BCR) signaling as judged by increased levels of phosphorylated Bruton's tyrosine kinase (pBtk), phosphorylated Spleen tyrosine kinase (pSyk), and nuclear receptor Nur77. Low dosage treatment with the pBtk inhibitor Ibrutinib reversed the altered B cell development in the spleen of sIgM -/- mice, suggesting that sIgM regulate splenic B cell differentiation by decreasing BCR signaling. Mechanistically, we show that B cells, which express BCRs specific to hen egg lysozyme (HEL) display diminished responsiveness to HEL stimulation in presence of soluble anti-HEL IgM antibodies. Our data identify sIgM as negative regulators of BCR signaling and suggest that they can act as decoy receptors for self-antigens that are recognized by membrane bound BCRs.

NUR77 inhibits the expression of TIMP2 and increases the migration and invasion of HTR-8/SVneo cells induced by CYR61.

OBJECTIVE: To investigate the function and mechanism of CYR61 on the migration and invasion of the trophoblast cell line, HTR-8/SVneo cells. STUDY DESIGN: The mRNA and protein levels of NUR77 in the placentas of normal and preeclampsia (PE) women were evaluated using real-time PCR and Western blot, respectively. Paraffin-embedded tissues were processed for localization of NUR77 protein in placental villus by immunohistochemistry. HTR-8/SVneo cells were cultured in the presence of CYR61, Ad-NUR77 or a small interfering RNA for NUR77 (Ad-sinur77). The expression of NUR77 in the HTR-8/SVneo cells was detected and the effects of CYR61 on the migration and invasion of HTR-8/SVneo cells were assessed in wound-healing and transwell experiments, respectively. Gelatin zymography was used to measure the MMP2 release in HTR-8/SVneo cells. RESULTS: NUR77 is significantly decreased in the placenta of women with PE compared with the levels during a normal pregnancy. CYR61 can significantly increase the expression of NUR77 in HTR-8/SVneo cells. CYR61, as well as NUR77, can promote HTR-8/SVneo cells migration and invasion, which can be blocked by Ad-sinur77. Both CYR61 and Ad-nur77 reduced the mRNA expression of TIMP2 in HTR-8/SVneo cells. CONCLUSIONS: CYR61 may promote HTR-8/SVneo cells migration and invasion through the upregulation of NUR77, leading to the increase of MMP2 release and the downregulation of TIMP2 expression.

Increased expression of NOR-1 mRNA in the skeletal muscles of cold-exposed neonatal chicks.

Nuclear receptor subfamily 4, group A (NR4A) subgroup orphan receptors are rapidly induced by various physiological stimuli and have been suggested to regulate oxidative metabolism and muscle mass in mammalian skeletal muscle. The results showed that the NR4A subgroup orphan receptor, NOR-1 (NR4A3), was acutely increased in skeletal muscles of neonatal chicks in response to short-term cold exposure. The increased NOR-1 gene expression was concomitant with cold-induced changes in gene expression of both myostatin and proliferator-activated receptor-gamma coactivator-1 (PGC-1α), and the increase in skeletal muscle mass. These observations suggest that NOR-1 might play a role in controlling skeletal muscle growth in cold-exposed neonatal chicks.

Nr4a1-eGFP is a marker of striosome-matrix architecture, development and activity in the extended striatum.

Transgenic mice expressing eGFP under population specific promoters are widely used in neuroscience to identify specific subsets of neurons in situ and as sensors of neuronal activity in vivo. Mice expressing eGFP from a bacterial artificial chromosome under the Nr4a1 promoter have high expression within the basal ganglia, particularly within the striosome compartments and striatal-like regions of the extended amygdala (bed nucleus of the stria terminalis, striatal fundus, central amygdaloid nucleus and intercalated cells). Grossly, eGFP expression is inverse to the matrix marker calbindin 28K and overlaps with mu-opioid receptor immunoreactivity in the striatum. This pattern of expression is similar to Drd1, but not Drd2, dopamine receptor driven eGFP expression in structures targeted by medium spiny neuron afferents. Striosomal expression is strong developmentally where Nr4a1-eGFP expression overlaps with Drd1, TrkB, tyrosine hydroxylase and phospho-ERK, but not phospho-CREB, immunoreactivity in "dopamine islands". Exposure of adolescent mice to methylphenidate resulted in an increase in eGFP in both compartments in the dorsolateral striatum but eGFP expression remained brighter in the striosomes. To address the role of activity in Nr4a1-eGFP expression, primary striatal cultures were prepared from neonatal mice and treated with forskolin, BDNF, SKF-83822 or high extracellular potassium and eGFP was measured fluorometrically in lysates. eGFP was induced in both neurons and contaminating glia in response to forskolin but SKF-83822, brain derived neurotrophic factor and depolarization increased eGFP in neuronal-like cells selectively. High levels of eGFP were primarily associated with Drd1+ neurons in vitro detected by immunofluorescence; however ∼15% of the brightly expressing cells contained punctate met-enkephalin immunoreactivity. The Nr4a1-GFP mouse strain will be a useful model for examining the connectivity, physiology, activity and development of the striosome system.

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.

Induction and intracellular localization of Nur77 dictate fenretinide-induced apoptosis of human liver cancer cells.

Fenretinide, a synthetic retinoid, is known to induce apoptosis in various cancer cells. However, the mechanism by which fenretinide induces apoptosis remains unclear. The current study examines the mechanisms of fenretinide-induced apoptosis in human hepatoma cells. The induction of Nur77 and the cytoplasmic distribution of Nur77 induced by fenretinide were positively correlated with the apoptotic effect of fenretinide in HCC cells. The sensitivity of Huh-7 cells was related to Nur77 translocation and targeting mitochondria, whereas the mechanism of resistance for HepG2 cells seemed due to Nur77 accumulating in the nucleus. The intracellular location of Nur77 was also associated with the differential capability of fenretinide-induced ROS generation in these two cell lines. In addition, the knockdown of Nur77 expression by siRNA greatly reduced fenretinide-induced apoptosis and cleaved caspase 3 in Huh-7 cells. Therefore, our findings demonstrate that fenretinide-induced apoptosis of HCC cells is Nur77 dependent and that the intracellular localization of Nur77 dictates the sensitivity of the HCC cells to fenretinide-induced apoptosis.