NR4A1 mRNA Research Articles

Role of local muscle contractile activity in the exercise-induced increase in NR4A receptor mRNA expression

Exercise upregulates the expression of NR4A receptors, which are involved in regulation of glucose and fatty acid utilization genes in skeletal muscle. The aims of our study were 1) to determine the role of local contractile activity on NR4A mRNA expression in skeletal muscle during exercise; and 2) to elucidate the mechanisms underlying the induction of NR4A mRNA expression in response to muscle contractile activity. Rats were subjected to an acute 3-h low-intensity swimming or a 3-h low-intensity treadmill running as a model of endurance exercise. Low-intensity swimming increased NR4A1 and NR4A3 mRNA in triceps but not in soleus muscle. Conversely, low-intensity treadmill running increased NR4A1 and NR4A3 mRNA in soleus but not in triceps muscle. NR4A mRNA increased concomitantly with reduced postexercise muscle glycogen, suggesting that gene expression of NR4A receptors occurs in muscles recruited during exercise. Furthermore, in resting rats, an acute 1-h local electrical stimulation of a motor nerve to the tibialis anterior muscle caused increases in NR4A1 and NR4A3 mRNA relative to the contralateral control muscle of the same animals. On the other hand, after 6 h of hindlimb immobilization, NR4A1 and NR4A3 mRNA were reduced in immobilized soleus muscle relative to contralateral control muscle. In addition, both NR4A1 and NR4A3 mRNA in epitrochlearis muscle were increased after 6-h incubation with 0.5 mM 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside, which activates AMP-activated protein kinase. These results suggest that 1) local muscle contractile activity is required for increased expressions of NR4A1 and NR4A3 mRNA during exercise; and 2) muscle contractile activity-induced increases in NR4A1 and NR4A3 mRNA may be mediated by AMPK activation, at least in part.

Characterisation of a novel NR4A2 mutation in Parkinson's disease brain

Objective: We performed a mutation screen of NR4A2 (also known as NURR1) in 409 Parkinson's disease (PD) patients. We identified a novel single base substitution in the 5′UTR of the NR4A2 (also known as NURR1) gene (c.-309C>T). Results: We have performed expression studies in neuronal cell lines showing that the c.-309C>T mutation reduces NR4A2 mRNA expression in vitro. We have confirmed this finding in vivo by performing allele specific real-time PCR from brain tissue harbouring the 309C>T mutation and show a 3.48±1.62 fold reduction in mRNA expression of the mutant allele compared to wild-type. In addition we have undertaken genome wide expression analysis of the mutant NR4A2 brain and shown underexpressed genes were significantly enriched for gene ontology categories in nervous system development and synaptic transmission and overexpressed genes were enriched for unfolded protein response and morphogenesis. Lastly we have shown that the c.-309C>T mutation abrogates the protective effect of wild-type NR4A2 against apoptopic stress. Conclusions: Our findings indicate the c.-309C>T mutation reduces NR4A2 expression resulting in the downregulation of genes involved in the development and maintenance of the nervous system and synaptic transmission. These downregulated pathways contained genes known to be transactivated by NR4A2 and were not disrupted in idiopathic PD brain suggesting causality of the mutation.

247 CANDIDATE mRNA REGULATING MEIOTIC RESUMPTION IN BOVINE CUMULUS-OOCYTE COMPLEXES

When cumulus–oocyte complexes (COC) are cultured with follicle-stimulating hormone (FSH), germinal vesicle breakdown (GVBD) occurs and is transcriptionally dependent. In mice, expression of mRNA for nuclear receptor subfamily 4, member 1 (Nr4A1), and early growth response 1 (Egr1) is reduced when GVBD is blocked by the transcriptional inhibitor DRB. The objectives of this study were to define the period required for transcription initiation in bovine COC, determine the pattern of expression for Nr4A1 and Egr1 mRNAs in bovine COC, and reduce Nr4A1 mRNA expression using small interfering RNA (siRNA) to determine the effect on GVBD. In Experiment 1, pools of COC were randomly assigned to mature in TCM-199 supplemented with 10% estrous cow serum, 1 μg mL–1 estradiol, 200 nm pyruvate, 5 μg mL–1 FSH, and 50 μg mL–1 gentamicin. The DRB (120 μm) was added at 0, 30, 60, 90, 120, 150, or 180 min of culture (n = 27 ± 2 COC/treatment per replication; n = 5 replications). The COC were harvested at 20 h and assessed for meiotic stage. In Experiment 2, COC were randomly assigned to maturation medium with FSH and were harvested at 0, 30, 60, 90, and 180 min of culture. Semiquantitative RT-PCR was used to assess Nr4A1, Egr1, and GAPDH (housekeeper) mRNA levels (n = 39 ± 2 COC/treatment per replication; n = 10 replications). In Experiment 3a, COC were matured for 30 min with either FSH, FSH + DRB, or FSH + 25 nm, 50 nm, or 100 nm siRNA for bovine Nr4A1 (siNr4A1). Relative levels of Nr4A1 mRNA were assessed (58 ± 4 COC/treatment per replication; n = 4 replications). In Experiment 3b, COC were matured 9 h in medium containing either FSH, FSH + DRB, FSH + 50 nm siNr4A1, or FSH + 50 nm nonspecific siRNA (siNS). Oocytes were assessed for meiotic stage (n = 13 ± 1 COC/treatment per replication; n = 4 replications). Data were analyzed by one-way ANOVA and Duncan’s test. For Experiment 1, when DRB was added after the start of culture, progressively more oocytes underwent GVBD (least squares mean ± SEM; 99 ± 7%A, 6 ± 7%E, 33 ± 7%D, 64 ± 7%C, 77 ± 7%B,C, 91 ± 7%A,B, 87 ± 7%A,B, 90 ± 7%A,B for control, 0 + DRB, 30 + DRB, 60 + DRB, 90 + DRB, 120 + DRB, 150 + DRB, 180 + DRB; A,B,C,D,EP < 0.05). Thus, the gene transcription required for GVBD occurred between 0 and 60 min of culture. For Experiment 2, relative levels of Nr4A1 mRNA increased (P < 0.05) at 30 min of culture (100 ± 18% v. 192 ± 18%, for 0 v. 30 min). Relative levels of Egr1 mRNA did not change during culture. For Experiment 3a, the relative expression of Nr4A1 mRNA was decreased in cultures containing siNr4A1 (100 ± 6%A, 32 ± 6%D, 62 ± 6%C, 59 ± 6%C,D, 86 ± 6%B for FSH, FSH + DRB, FSH + 25 nm siNr4A1, FSH + 50 nm siNr4A1, FSH + 100 nm siNr4A1; A,B,C,DP < 0.05). For Experiment 3b, there were no differences in the proportions of COC undergoing GVBD at 9 h of culture in the FSH and FSH + siNS treatments. There was a decreased percentage of GVBD oocytes at 9 h of culture with either DRB or 50 nm siNr4A1 (95 ± 4%A, 4 ± 4%C, 65 ± 4%B, 97 ± 4%A for FSH, FSH + DRB, FSH + 50 nm siNr4A1, FSH + 50 nm siNS; A,B,CP < 0.05). These data are consistent with the hypothesis that Nr4A1 may play a role in regulating GVBD in bovine COC. This research was supported by USDA-NRI Grant 35205-12810, NIH Grant 1 R03 HD043875, and the North Carolina Agricultural Research Service.

Nr4a2 is essential for the differentiation of dopaminergic neurons during zebrafish embryogenesis

Nr4a2 is a member of the orphan nuclear receptor gene superfamily, which has been found to be critical for the development and maintenance of mesencephalic dopaminergic (DA) neurons. To uncover the molecular mechanisms by which Nr4a2 contributes to the development of DA neurons, we have applied zebrafish to study the topographic distribution of nr4a2b transcripts, as well as its correlation with neuronal progenitor marker ( neurogenin 1) and DA neuron markers ( tyrosine hydroxylase, TH and DA transporter, DAT) during neurogenesis. Our studies showed that although nr4a2b transcripts did not co-localize with TH and DAT transcripts in the posterior tuberculum (PT area), knockdown of Nr4a2 resulted in a significant decrease of TH + and DAT + DA neurons in the PT area, accompanied by a reduction of DA transmitter, which were partially rescued by the injection of mouse Nr4a2 mRNA. Surprisingly, the number of nr4a2b + cells in Nr4a2-deficient embryos was increased by 1.6 fold. These results suggest that Nr4a2 may play an important role in the differentiation and maturation rather than the survival of DA progenitors in the PT area during zebrafish early embryogenesis.

Persistent alterations in mesolimbic gene expression with abstinence from cocaine self-administration.

Cocaine-responsive gene expression changes have been described after either no drug abstinence or short periods of abstinence. Little data exist on the persistence of these changes after long-term abstinence. Previously, we reported that after discrete-trial cocaine self-administration and 10 days of forced abstinence, incubation of cocaine reinforcement was observable by a progressive ratio schedule. The present study used rat discrete-trial cocaine self-administration and long-term forced abstinence to examine extinction responding, mRNA abundance of known cocaine-responsive genes, and chromatin remodeling. At 30 and 100 days of abstinence, extinction responding increased compared to 3-day abstinent rats. Decreases in both medial prefrontal cortex (mPFC) and nucleus accumbens c-fos, Nr4a1, Arc, and EGR1 mRNA were observed, and in most cases persisted, for 100 days of abstinence. The signaling peptides CART and neuropeptide Y (NPY) transiently increased in the mPFC, but returned to baseline levels following 10 days of abstinence. To investigate a potential regulatory mechanism for these persistent mRNA changes, levels of histone H3 acetylation at promoters for genes with altered mRNA expression were examined. In the mPFC, histone H3 acetylation decreased after 1 and 10 days of abstinence at the promoter for EGR1. H3 acetylation increased for NPY after 1 day of abstinence and returned to control levels by 10 days of abstinence. Behaviorally, these results demonstrate incubation after discrete-trial cocaine self-administration and prolonged forced abstinence. This incubation is accompanied by changes in gene expression that persist long after cessation of drug administration and may be regulated by chromatin remodeling.

Prostaglandin E2 Regulates the Nuclear Receptor NR4A2 in Colorectal Cancer

Many lines of research implicate cyclooxygenase 2-derived prostaglandins in tumor growth and metastasis. More specifically, we have shown that prostaglandin E2 (PGE2) promotes cell proliferation and invasion through transactivation of the epidermal growth factor receptor, initiates immune evasion through induction of decay accelerating factor, and transactivates peroxisome proliferator-activated receptor delta, leading to increased polyp size and multiplicity. We continue to identify novel PGE2 target genes in colorectal carcinoma cells and report here that an immediate early gene, nuclear factor NR4A2 (Nurr1), is induced by PGE2 that in turn regulates cell death. Originally described as a critical dopaminergic neuron growth factor receptor, NR4A2 expression is rapidly but transiently induced by PGE2 in a cAMP/protein kinase A-dependent manner. NR4A2 binds to the cognate NBRE response element and enhances transcription of a reporter construct in colorectal carcinoma cells. Furthermore, NR4A2 expression is elevated in Apc-/+ mouse adenomas and its levels were further increased following PGE2 treatment. Human colorectal cancers relative to matched normal mucosa showed increased NR4A2 expression. Although not previously described in epithelial tissues, NR4A2 protein localizes to proliferating crypts of Apc-/+ mouse intestine. Finally, functional studies reveal that PGE2-mediated protection from apoptosis is completely inhibited by a dominant-negative NR4A2 construct. Building on previous reports from our group on the peroxisome proliferator-activated receptor family of nuclear receptors, these most recent data suggest that NR4A2, a member of another family of nuclear receptors can stimulate progression of colorectal cancer downstream from cyclooxygenase 2-derived PGE2.

Mutations in NR4A2 associated with familial Parkinson disease.

NR4A2, encoding a member of nuclear receptor superfamily, is essential for the differentiation of the nigral dopaminergic neurons. To determine whether NR4A2 is a susceptibility gene for Parkinson disease, we carried out genetic analyses in 201 individuals affected with Parkinson disease and 221 age-matched unaffected controls. We identified two mutations in NR4A2 associated with Parkinson disease (-291Tdel and -245T-->G), which map to the first exon of NR4A2 and affect one allele in 10 of 107 individuals with familial Parkinson disease but not in any individuals with sporadic Parkinson disease (n = 94) or in unaffected controls (n = 221). The age at onset of disease and clinical features of these ten individuals were not different from those of individuals with typical Parkinson disease. The mutations resulted in a marked decrease in NR4A2 mRNA levels in transfected cell lines and in lymphocytes of affected individuals. Additionally, mutations in NR4A2 affect transcription of the gene encoding tyrosine hydroxylase. These data suggest that mutations in NR4A2 can cause dopaminergic dysfunction, associated with Parkinson disease.