Nur77 Protein Expression Research Articles

Exogenous α-Synuclein Induces Oxidative Damage to Dopaminergic Neurons Through p-NMDAR2B/Nur77.

Alpha-synuclein (α-syn) is a major pathological marker of Parkinson's disease (PD), and its abnormal expression and aggregation lead to dopaminergic neuron degeneration, in which oxidative stress plays an important role. However, the exact molecular mechanism by which α-syn causes PD remains unclear. In this study, exogenous α-syn, also known as α-syn preformed fibrils (α-syn PFFs), was used to construct in vivo and in vitro models of PD. Behavioral, Western blotting, biochemical, immunofluorescence, flow cytometry, electron microscopy, etc. were used to investigate the pathological mechanism of PD induced by α-syn. We found that 6months after striatum injection of α-syn PFFs, mice exhibited motor deficits. Meanwhile, the protein expression of pS129-α-syn (p-α-syn) and α-syn oligomer significantly increased, while the expression of TH significantly decreased, and the oxidative stress in the substantia nigra was aggravated. In addition, we found an increase in the protein expression of NMDAR2B and p-Tyr1472-NMDAR2B (p-NMDAR2B) and a decrease in the protein expression of Nur77. However, in α-syn PFFs-induced SH-SY5Y cells, we found that inhibiting p-NMDAR2B increased the protein expression of Nur77, while overexpression of Nur77 did not affect the expression of p-NMDAR2B. Inhibition of p-NMDAR2B and overexpression of Nur77 reversed α-syn PFF-induced oxidative stress, thus reducing mitochondrial damage and cytotoxicity. Therefore, we speculate that α-syn PFF-induced oxidative stress in dopaminergic neurons may be mediated by p-NMDAR2B/Nur77. Our study provides novel insights into the pathology mechanism underlying α-syn-induced PD.

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.

Nur77 suppression facilitates androgen deprivation-induced cell invasion of prostate cancer cells mediated by TGF-β signaling.

Androgen deprivation therapy (ADT) remains a standard treatment for advanced prostate cancers. However, recent studies revealed that while inhibiting the growth of certain types of prostate cancer cells, ADT promotes invasion. In the current study, we explored the effects of Nur77, an orphan nuclear receptor, on prostate cancer cell invasion following ADT. Androgen receptor (AR) and Nur77 protein expression in patient tissues and cell lines were quantified via ELISA and western blot. The effects of AR-signaling on Nur77 expression were examined. The effects of Nur77 over-expression and knockdown on ADT-induced prostate cancer cell invasion were characterized. The results showed that AR and Nur77 are both highly expressed in prostate cancers of patients. Nur77 is positively regulated by AR-signaling at transcriptional level in NCI-H660, a widely used prostate cancer cell line. AR antagonists, Casodex and MDV3100 treatment resulted in significant inhibition of prostate cancer cell growth but enhanced cancer cell invasion. Nur77 over-expression blocked invasion-promoting effect of ADT, which is consistent with the down-regulation of MMP9 and Snail protein expression. Further mechanistic investigations showed that Nur77 inhibited transcription of TGF-β target genes (Snail and MMP9), and thereby inhibits TGF-β-mediated prostate cancer cell invasion following androgen antagonism. In addition, our data suggested the nature of this inhibitory effect of Nur77 on TGF-β-signaling is selective, for Smad3-signaling, the classical effector of TGF-β-signaling, was not interrupted by Nur77 over-expression. Considering the limited success of management of prostate cancer metastasis following ADT, our data strongly suggest that Nur77 regulation could be a promising direction for search of complementary therapeutic strategy on top of classic ADT therapy.

Endogenous Nur77 Is a Specific Indicator of Antigen Receptor Signaling in Human T and B Cells.

Distinguishing true Ag-stimulated lymphocytes from bystanders activated by the inflammatory milieu has been difficult. Nur77 is an immediate early gene whose expression is rapidly upregulated by TCR signaling in murine T cells and human thymocytes. Nur77-GFP transgenes serve as specific TCR and BCR signaling reporters in murine transgenic models. In this study, we demonstrate that endogenous Nur77 protein expression can serve as a reporter of TCR and BCR specific signaling in human PBMCs. Nur77 protein amounts were assessed by immunofluorescence and flow cytometry in T and B cells isolated from human PBMCs obtained from healthy donors that had been stimulated by their respective Ag receptors. We demonstrate that endogenous Nur77 is a more specific reporter of Ag-specific signaling events than the commonly used CD69 activation marker in both human T and B cells. This is reflective of the disparity in signaling pathways that regulate the expression of Nur77 and CD69. Assessing endogenous Nur77 protein expression has great potential to identify Ag-activated lymphocytes in human disease.

Amelioration of palmitate-induced metabolic dysfunction in L6 muscle cells expressing low levels of receptor-interacting protein 140.

We have shown that reduced expression of receptor-interacting protein 140 (RIP140) alters the regulation of fatty-acid (FA) oxidation in muscle. To determine whether a high level of FA availability alters the effects of RIP140 on metabolic regulation, L6 myotubes were transfected with or without RNA interference oligonucleotide sequences to reduce RIP140 expression, and then incubated with high levels of palmitic acid, with or without insulin. High levels of palmitate reduced basal (53%-58%) and insulin-treated (24%-44%) FA uptake and oxidation, and increased basal glucose uptake (88%). In cells incubated with high levels of palmitate, low RIP140 increased basal FA uptake and insulin-treated FA oxidation and glucose uptake, and decreased basal glucose uptake and insulin-treated FA uptake. Under basal conditions, low RIP140 increased the mRNA content of FAT/CD36 (159%) and COX4 (61%), as well as the protein content of Nur77 (68%), whereas the mRNA expression of FGF21 (50%) was decreased, as was the protein content of CPT1b (35%) and FGF21 (44%). Under insulin-treated conditions, low RIP140 expression increased the mRNA content of MCAD (84%) and Nur77 (84%), as well as the protein content of Nur77 (23%). Thus, a low level of RIP140 restores the rates of FA uptake in the basal state, in part via a reduction in upstream insulin signaling. Our data also indicate that the protein expression of Nur77 may be modulated by RIP140 when muscle cells are metabolically challenged by high levels of palmitate.

Haloperidol-induced Nur77 expression in striatopallidal neurons is under the control of protein phosphatase 1 regulation by DARPP-32

Impaired dopaminergic signaling in the striatum is involved in diseases as diverse as Parkinson's disease, addiction, and schizophrenia. An important pathophysiological aspect is the loss of balance between striatopallidal and striatonigral pathways. Nur77 is an orphan nuclear receptor and dopamine-regulated immediate-early gene. Classical antipsychotic drugs widely used in the treatment of schizophrenia, such as haloperidol, increase Nur77 mRNA expression in the striatum. However, little is known about the intracellular signaling pathways involved in Nur77 induction. Here, using pharmacological approaches and transgenic mutant mice, we investigated the mechanisms underlying the up-regulation of Nur77 protein expression in the dorsal striatum after haloperidol injection. In drd1a::EGFP transgenic mice that express GFP in D1 neurons, Nur77 up-regulation induced by haloperidol occurred predominantly in GFP-negative neurons. In Gαolf heterozygous mutant mice, in which cAMP production in response to A2A stimulation is impaired in the striatum, haloperidol effect was not altered. In contrast, in DARPP-32 knock-in mutant mice bearing a T34A point mutation of the site responsible for cAMP-dependent phosphatase 1 inhibition, Nur77 up-regulation by haloperidol was prevented. Haloperidol also induced Nur77 protein in D2 neurons of the nucleus accumbens core of wild type but not T34A knock-in mice. Thus, our results show that expression of Nur77 is induced by haloperidol in D2 receptors-expressing medium-sized spiny neurons, through cAMP-dependent regulation of protein phosphatase 1, which is likely to modulate the effects of other protein kinases. Our results clarify the mechanisms of Nur77 induction by antipsychotic and its possible contribution to extrapyramidal effects.

Antiproliferative Cardiac Glycosides from the Latex of Antiaris toxicaria

Phytochemical investigation of the latex of Antiaris toxicaria resulted in the isolation of 15 new [antiarosides J-X (1-15)] and 17 known cardiac glycosides. The effects of the cardiac glycosides on apoptosis and the expression of orphan nuclear receptor Nur77 were examined in human NIH-H460 lung cancer cells. Several of the cardiac glycosides induced apoptosis in lung cancer cells, which was accompanied by induction of Nur77 protein expression. Treatment of cancer cells with the cardiac glycosides resulted in translocation of the Nur77 protein from the nucleus to the cytoplasm and subsequent targeting to mitochondria. The results show that the cardiac glycosides exert their apoptotic effect through the Nur77-dependent apoptotic pathway.

The Orphan Nuclear Receptor Nur77 Suppresses Endothelial Cell Activation Through Induction of IκBα Expression

Endothelial inflammation plays a critical role in the development and progression of cardiovascular disease, albeit the mechanisms need to be fully elucidated. Nur77 is highly expressed in vascular endothelial cells (ECs) and plays a role in the regulation of cell proliferation and angiogenesis; its role in vascular inflammation, however, remains unknown. Treatment of human umbilical vein ECs (HUVECs) with tumor necrosis factor (TNF)-alpha substantially increased the transcription and protein expression of Nur77 in a dose and time-dependent manner, as determined by Northern blot and Western blot analysis. Adenovirus mediated overexpression of Nur77 markedly increased the intracellular levels of IkappaBalpha by approximately 4-fold, whereas overexpression of dominant negative Nur77 (DN-Nur77), which lacks its transactivation domain, had no effect on IkappaBalpha expression, suggesting that Nur77 is an important transcriptional factor in controlling IkappaBalpha expression in ECs. Furthermore, overexpression of Nur77 significantly increased IkappaBalpha promoter activity via directly binding to a Nur77 response element in the IkappaBalpha promoter. Importantly, overexpression of Nur77, but not DN-Nur77, protected ECs against the TNF-alpha- and interleukin-1beta-induced endothelial activation, as characterized by attenuation in the nuclear factor kappaB activation, expression of adhesion molecules ICAM-1 and VCAM-1, and monocytic adherence to ECs. These results indicate that Nur77 negatively regulates the TNF-alpha- and interleukin-1beta-induced vascular EC activation by transcriptionally upregulation of IkappaBalpha expression.

Depletion of HDAC7 and de-repression of Nur77: a mechanism for sensitivity of cutaneous lymphoma (CTCL) cells to pan- histone deacetylase inhibitor Panobinostat (LBH589)

14542 Background: HDAC7 belongs to the class IIA histone deacetylases (HDACs) and is overexpressed in thymocytes, where it transcriptionally represses the proapoptotic nuclear orphan receptor Nur77. Following an apoptotic stimulus, Nur77 translocates from the nucleus to mitochondria, where it binds and converts Bcl-2 into a pro-apoptotic protein, allowing caspase activation and apoptosis. Panobinostat (P) (Novartis Pharmaceuticals) has a high level of clinical activity against advanced CTCL cells. However, the molecular basis of this activity remains unclear. Methods: RT-PCR, confocal immunofluorescent microscopy and immunoblot analyses were utilized to determine the effects of P on the levels and localization of HDAC7 and Nur77 in cultured CTCL HH and HUT78 cells. Apoptosis was determined by the morphologic features and by estimating the sub-G1 hypodiploid DNA-containing fraction of cells. The sensitizing effects of the ectopic overexpression of Nur77 and its knockdown by Nur77 siRNA on P-induced apoptosis were also determined. Results: Treatment with P (5 to 20 nM) rapidly (4 hours) depleted the in vitro and intracellular activity, as well as depleted the mRNA and protein levels of HDAC7, but simultaneously induced the mRNA and protein expression of Nur77 in human CTCL HH and HUT-78 cells. P induced Nur77 translocation from the nucleus to mitochondria. This was associated with apoptosis of HH and HUT-78 cells. Ectopic overexpression of Nur77 induced apoptosis and sensitized HH cells to P. Conversely, knockdown of Nur77 levels inhibited P-induced apoptosis of HH cells. Co-treatment with ABT-737, a Bcl-2/BclxL antagonist (0,1 to 1.0 uM) significantly increased P-induced apoptosis of CTCL cells. In 5 samples of primary CTCL cells, ex-vivo treatment with P also dose-dependently increased the % of non-viable cells to up to 35 to 82%. Conclusions: These findings mechanistically implicate the role of HDAC7 down regulation and resulting de-repression and mitochondrial translocation of Nur77 in sensitizing human CTCL cells to P-induced cell death. These results also indicate that co-treatment with an anti-Bcl-2 agent e.g., a BH3 domain-only mimetic, would augment the anti-CTCL activity of P. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis Institute for Biomedical Research Inc. Novartis Institute for Biomedical Research Inc. Merck & Co., Novartis, Novartis Institute for Biomedical Research Inc.

Effects of retinoid X receptor activation on lightly oxidized low-density lipoprotein induced cell proliferation of macrophages: experiment with murine RAW264.7 cells

Objective To investigate the effects and mechanism of retinoid X receptor (RXR) activation on macrophages proliferation induced by lightly oxidized low-density lipoprotein (LoxLDL).Methods Serum-starved mouse macrophages of the line RAW264. 7 were stimulated with 20 μg/mLLoxLDL for 24 h to induce proliferation in the absence or presence of varying doses of RXR special agonist 9-cis retinoid acid (RA). The cell viability was assayed by MTT method. The DNA synthesis was assayed by BrdU ELISA. The apoptotic percentage of cells was measured by flow cytometry using propidium iodide (PI) staining. Nur77 and RARβ protein expressions were detected by Western blotting. Results After treated by LoxLDL for 24 h, the cell viability and DNA synthesis ( A value) of the RAW264.7 ceils were increased by 0.79±0.12 and 1.81±0.31 respectively. Co-incubated with LoxLDL and 10-s mol/L and 10-7 mol/L 9-cisRA the ceil viability decreased to 0.43±0.10 and 0.26±0.07 respectively( both P＜0. 05 ), and the DNA synthesis decreased to 1.14±0.43 and 0.72±0.06 respectively(both P＜0.05 ) The apoptotic rate of the ceils treated with LoxLDL was (3.08±0.30) %, not significantly different from those of the cells cocultured with LoxLDL and 10-9-10-7 mol/L 9-cisRA (2.74±0.13 ) % , (2.94±0.24) % , and (2.48±0.42) % respectively, all P＞0.05. The orphan nuclear receptor Nur77 protein expression was up-regulated by more than 5 times after LoxLDL treatment. 9-cisRA down-regulates the Nut77 expression and increased the expression of the downstream gene RARβ. Condnsion RXR activation significantly inhibits the macrophage proliferation induced by LoxLDL. The mechanism may be related to the regulation of RXR/Nur77 heterodimer and its downstream gene RARβ. Key words: Coronary arteriosclerosis ; Lipoprotein, LDL; Macrophages ; Retinoid X receptor

Mono-(2-ethylhexyl) phthalate (MEHP) induces nuclear receptor 4A subfamily in NCI-H295R cells: A possible mechanism of aromatase suppression by MEHP

Phthalate esters are widely used as plasticizers for polyvinylchloride and are suspected of functioning as endocrine disrupters. Di-(2-ethylhexyl) phthalate (DEHP), the most important phthalate ester in commercial use, has been reported to act as a rodent reproductive toxicant. In the present study, we investigated the effects of phthalate esters on aromatase (CYP19) activity and on its gene expression in a human adrenocortical carcinoma cell line, NCI-H295R. Mono-(2-ethylhexyl) phthalate (MEHP), a principle metabolite of DEHP, dose-dependently suppressed aromatase activity and its transcription level. Furthermore, MEHP rapidly and transiently induced transcription of the genes which encode nuclear receptor 4A subfamily members (Nur77, Nurr1 and NOR-1), and up-regulated Nur77 promoter activation and Nur77 protein expression in the cells. MEHP-induced Nur77 transcription was inhibited by bisindolylmaleimide I (protein kinase C inhibitor) and wortmannin (phosphoinositide 3-kinase inhibitor). Finally, ectopic expression of Nur77 markedly suppressed forskolin-induced transcriptional activation of promoters I.3 and II of the CYP19 gene. These results suggest that the suppression of aromatase activity and its transcription level by MEHP exposure to NCI-H295R cells was regulated through the rapid and transient expression of Nur77 gene.

Modulation of steroidogenic enzymes by orphan nuclear transcriptional regulation may control diverse production of cortisol and androgens in the human adrenal.

The capacity of the adrenal to produce cortisol is controlled in part by 21-hydroxylase (CYP21) and the production of androgens by 17-hydroxylase/17-20-lyase (CYP17), in response to secretagogues including ACTH, angiotensin-II (A-II) and insulin. In this study we examined the capacity of human adrenocortical cells to produce cortisol and androgens in response to these secretagogues and their ability to regulate the expression of CYP21 and CYP17. In H-295 cells, forskolin and A-II were found to stimulate production of cortisol relative to androstenedione and a similar pattern of steroid production was noted in primary human adrenocortical cells. Both mRNA and protein expression of CYP21 was upregulated with forskolin and A-II alone and in combination, as detected by Northern and Western blotting. Whereas expression of CYP17 mRNA and protein was up regulated in the presence of forskolin and forskolin in combination with insulin. The ability of steroidogenic factor-1 (SF-1) and nur77 to regulate transcription of these enzymes was examined. Forskolin, A-II and insulin increased the protein expression of SF-1. Increased binding of SF-1 to its response element in the presence of forskolin, A-II and insulin was observed. Nur77 was expressed primarily in the zona glomerulosa and fasciculata. Increased protein expression of nur77 and the greatest binding of nur77 to its response element was seen when cells were stimulated with A-II in combination with forskolin. These data indicate that nur77 may preferentially regulate steroid enzyme genes relevant to cortisol production and thereby regulate differential cortisol and adrenal androgen production.

Regulation of the Nuclear Orphan Receptor Nur77 in Bone by Parathyroid Hormone

Osteoblasts function under the control of several hormones and growth factors. Among them, parathyroid hormone (PTH) and steroid hormones have significant effects on bone metabolism. We show that PTH induced the expression of Nur77, a member of the NGFI-B subfamily of nuclear orphan receptors in bone. PTH rapidly and transiently induced Nur77 mRNA in primary mouse osteoblasts that peaked at 1 h and at 10 nM of hormone. Cycloheximide did not affect the induction of Nur77 mRNA, suggesting that protein synthesis is not required for the PTH effect. PTH also induced Nur77 mRNA in calvariae cultures. Finally Nur77 protein expression was induced in nuclear protein extracts of cells treated with PTH. NGFI-B nuclear receptors have been implicated in retinoic acid, vitamin D, and thyroid hormone signaling. We propose that induction of NGFI-B nuclear orphan receptors represents a potential cross-talk mechanism between PTH and steroid hormone signaling to regulate bone metabolism.

Costimulatory signals are required for induction of transcription factor Nur77 during negative selection of CD4(+)CD8(+) thymocytes.

A major question in end-stage T cell development is how T cell receptor(TCR) ligation on immature CD4(+)CD8(+) double positive thymocytes is translated into either survival (positive selection) or apoptotic (negative selection) signals. Because different types of antigen-presenting cells (APCs) induce positive or negative selection in the thymus and express different costimulatory molecules, involvement of such costimulatory molecules in determining cell fate of DP thymocytes is considered here. If TCR-generated signals are modulated by APCs, this should be reflected in the activation of distinct biochemical pathways. We here demonstrate that costimulatory signals involved in negative selection also are required for induction of protein expression of Nur77 and its family members. These transcription factors are critically involved in negative but not positive selection. In contrast, the signals that costimulate negative selection are not required for induction of several molecular events associated with positive selection. These include activation of the immediate early gene Egr-1, the mitogen-activated protein kinase ERK2, and surface expression of the CD69 marker. Thus, costimulation for negative selection selectively provides signals for activation of apoptotic mediators. These data provide molecular insights into how TCR-engagement by ligands on different thymic APCs can determine cell fate.

Protein kinase A serves as a primary pathway in activation of Nur77 expression by gonadotropin-releasing hormone in the LβT2 mouse pituitary gonadotroph tumor cell line

Nur77 belongs to a subfamily of nuclear receptors that includes two other members, Nor-1 and Nurr1. It plays an important role in a number of biological processes, including regulation of signaling functions in the hypothalamo-pituitary-adrenal axis, regulation of thymocyte apoptosis, regulation of steroidogenesis and regulation of tumor cell proliferation and apoptosis. In previous studies, using DNA microarray analysis of the effects of the gonadotropin-releasing hormone (GnRH) on the mouse pituitary gonadotroph cell line LbetaT2, we identified Nur77 as one of the highly regulated immediate early genes involved in this response, with >40-fold upregulation after 1 h of treatment of the cells with the GnRH agonist [D-Ala6GnRH (GnRHA)]. GnRH is a hypothalamic decapeptide that stimulates the secretion and expression of gonadotropins (follicle stimulating hormone, FSH and luteinizing hormone releasing hormone, LH) from anterior pituitary through activation of high affinity receptors present on cell membrane of pituitary gonadotropes. In addition to pituitary, the presence of GnRH high affinity receptors has been reported in various cancers and cancer cell lines. In addition, GnRH and its analogs are clinically used in the treatment of prostate cancer. To elucidate the molecular mechanism involved in regulation of Nur77 by GnRH, we first confirmed upregulation of Nur77 in response to GnRH analog (GnRHA) in LbetaT2 cells. Nur77 mRNA was upregulated within 30 min of GnRHA treatment and returned to nearly basal level after 24 h of treatment. Nur77 protein expression was upregulated after 2 h of treatment and remained steady even after 12 h of treatment. The expression of Nur77 mRNA was induced by GnRHA in a dose-dependent manner. Induction of Nur77 expression was stimulated on treatment of cells with forskolin and 8-Br-cAMP, whereas H-89, a specific inhibitor of PKA pathway significantly inhibited GnRHA-induced Nur77 expression. Treatment of cells with both H-89 and EGTA completely blocked the GnRHA-induced expression of Nur77, indicating that both calcium and cAMP/PKA play an important role in regulation of Nur77 expression by GnRHA. Analysis of the protein kinase C (PKC) signaling pathway using specific inhibitors for PKC, Erk1/2, p38 and JNK demonstrated that these pathways are not involved in GnRHA-induced Nur77 expression. Based on our results, we conclude that activation of protein kinase A is the major mechanism regulating the expression of Nur77 by GnRH which may serve as a down-stream signaling gene to mediate the antitumor effects of GnRH.