NR5A1 Gene Research Articles

NR4A1 in marginal zone B cells decreases atherosclerosis

Background and Aims: Marginal zone B (MZB) cells protect from atherosclerosis by regulating the T cell follicular helper (Tfh)/ germinal centre (GC) response, in part through upregulation of PDL1 expression (1). NR4A genes are among the most highly induced genes in B cells following BCR stimulation and we previously found that NR4A1 was significantly up-regulated in MZB cells after the consumption of high fat/ high cholesterol (HF/HC) diet (HF/HC diet) in mice. However, the role of NR4A1 in this context is still unknown.

Epinephrine May Contribute to the Persistence of Traumatic Memories in a Post-traumatic Stress Disorder Animal Model.

The importance of catecholamines in post-traumatic stress disorder (PTSD) still needs to be explored. We aimed to evaluate epinephrine’s (EPI) causal role and molecular mechanism for the persistence of PTSD traumatic memories. Wild-type (WT) and EPI-deficient mice (phenylethanolamine-N-methyltransferase-knockout mice, Pnmt-KO) were induced with PTSD and behavioral tests were performed. Some Pnmt-KO mice were administered with EPI or vehicle. Catecholamines were quantified by HPLC-ED. Nr4a1, Nr4a2, and Nr4a3 mRNA expression were evaluated by real-time PCR in hippocampus samples. It was observed an increase in EPI and freezing behavior, and a decrease in open arm entries in the elevated plus-maze test and time spent in the light in the light–dark test in WT mice in the PTSD-induction group compared to control. After induction of PTSD, Pnmt-KO mice showed a decrease in freezing, as well as an increase in open arm entries and transitions between compartments compared to WT. After PTSD induction, Pnmt-KO mice administered with EPI showed an increase in freezing compared with the vehicle. On day 0 of PTSD induction, it was observed an increase in mRNA expression of Nr4a2 and Nr4a3 genes in the hippocampus of WT mice compared to control, contrary to Pnmt-KO mice. In conclusion, our data suggest that EPI may be involved in the persistence of traumatic memories in PTSD, possibly through enhancement of the expression of Nr4a2 and Nr4a3 genes in the hippocampus. Peripheral administration of EPI restored contextual traumatic memories in Pnmt-KO mice, which suggests a causal role for EPI. The persistence of contextual traumatic memories may contribute to anxiety-like behavior and resistance of traumatic memory extinction in this PTSD mice model.

Screening for structural variants of four candidate genes in dogs with disorders of sex development revealed the first case of a large deletion in NR5A1

Disorders of sex development (DSD) are important causes of infertility and sterility, and are risk factors for gonadal carcinogenesis. Many DSDs are caused by genetic factors, mainly sex chromosome abnormalities or mutations of genes involved in sexual development, as well as structural variants (SVs) - large deletions, duplications, and insertions, if these overlap genes involved in sex development. The aim of this study was to determine if there were SVs in four candidate genes - NR0B1 (DAX1), NR5A1, RSPO1, and SOX3 - using droplet digital PCR (ddPCR). There was study of two cohorts of dogs with DSD, including 55 animals with XX DSD and 15 with XY DSD. In addition, 40 control females and 10 control males were included in the study. Among cases, for which there were evaluations, a large deletion consisting of four exons of the NR5A1 gene was identified in a Yorkshire Terrier with a rudimentary penis, hypospadias, bilateral cryptorchidism, and spermatogenesis inactive testes. This is the first mutation in the NR5A1 gene leading to XY DSD phenotype to be reported in domestic animals. There were no SVs in the genes evaluated in the present study in the cohort of dogs with XX DSD. The results from this study provide evidence that the large structural variants of these genes are rarely associated with the DSD phenotype in dogs.

Clinical Evaluation of a Custom Gene Panel as a Tool for Precision Male Infertility Diagnosis by Next-Generation Sequencing.

Background: Up to 15% of couples are infertile and male factor infertility accounts for approximately 50% of these cases. Male infertility is a multifactorial pathological condition. The genetic of male infertility is very complex and at least 2000 genes are involved in its etiology. Genetic testing by next-generation sequencing (NGS) technologies can be relevant for its diagnostic value in male infertile patients. Therefore, the aim of this study was to implement the diagnostic offer with the use of an NGS panel for the identification of genetic variants. Methods: We developed an NGS gene panel that we used in 22 male infertile patients. The panel consisted of 110 genes exploring the genetic causes of male infertility; namely spermatogenesis failure due to single-gene mutations, central hypogonadism, androgen insensitivity syndrome, congenital hypopituitarism, and primary ciliary dyskinesia. Results: NGS and a subsequent sequencing of the positive pathogenic or likely pathogenic variants, 5 patients (23%) were found to have a molecular defect. In particular, pathogenic variants were identified in TEX11, CCDC39, CHD7, and NR5A1 genes. Moreover, 14 variants of unknown significance and 7 novel variants were found that require further functional studies and family segregation. Conclusion: This extended NGS-based diagnostic approach may represent a useful tool for the diagnosis of male infertility. The development of a custom-made gene panel by NGS seems capable of reducing the proportion of male idiopathic infertility.

Prevalence of gene mutations in a Chinese 46,XY disorders of sex development cohort detected by targeted next-generation sequencing.

46,XY disorders of sex development (DSD) is characterized by incomplete masculinization genitalia, with gonadal dysplasia and with/without the presence of Müllerian structures. At least 30 genes related to 46,XY DSD have been found. However, the clinical phenotypes of patients with different gene mutations overlap, and accurate diagnosis relies on gene sequencing technology. Therefore, this study aims to determine the prevalence of pathogenic mutations in a Chinese cohort with 46,XY DSD by the targeted next-generation sequencing (NGS) technology. Eighty-seven 46,XY DSD patients were enrolled from the Peking Union Medical College Hospital (Beijing, China). A total of fifty-four rare variants were identified in 60 patients with 46,XY DSD. The incidence of these rare variants was approximately 69.0% (60/87). Twenty-five novel variants and 29 reported variants were identified. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, thirty-three variants were classified as pathogenic or likely pathogenic variants and 21 variants were assessed as variants of uncertain significance. The overall diagnostic rate was about 42.5% based on the pathogenic and likely pathogenic variants. Androgen receptor (AR), steroid 5-alpha-reductase 2 (SRD5A2) and nuclear receptor subfamily 5 Group A member 1 (NR5A1) gene variants were identified in 21, 13 and 13 patients, respectively. The incidence of these three gene variants was about 78.3% (47/60) in patients with rare variants. It is concluded that targeted NGS is an effective method to detect pathogenic mutations in 46,XY DSD patients and AR, SRD5A2, and NR5A1 genes were the most common pathogenic genes in our cohort.

Clinical and molecular characteristics of patients with 46,XY DSD due to NR5A1 gene mutations

Steroidogenic factor 1 (SF1, NR5A1) is a nuclear receptor that regulates multiple genes involved in adrenal and gonadal development, steroidogenesis, and the reproductive axis. Human mutations in SF1 were initially found in patients with severe gonadal dysgenesis and primary adrenal failure. However, more recent case reports have suggested that heterozygous mutations in SF1 may also be found in patients with 46,XY partial gonadal dysgenesis and underandrogenization but normal adrenal function. We have analyzed the gene encoding SF1 (NR5A1) in a cohort of 310 Russian patients with 46,XY disorders of sex development (DSD). Heterozygous SF1 variants were found in 36 out of 310 (11.6%) of cases, among them 15 were not previously described. We have not found any phenotype-genotype correlations and any clinical and laboratory markers that would allow to suspect this type of before conducting molecular genetic analysis.

Oncogenic Orphan Nuclear Receptor NR4A3 Interacts and Cooperates with MYB in Acinic Cell Carcinoma.

Acinic cell carcinoma (AcCC) is a morphologically distinctive salivary gland malignancy often associated with chromosome rearrangements leading to overexpression of the NR4A3 transcription factor. However, little is known about how NR4A3 contributes to AcCC biology. Detailed RNA-sequencing of 21 archived AcCC samples revealed fusion reads arising from recurrent t(4;9), t(9;12), t(8;9) or t(2;4) chromosomal translocations, which positioned highly active enhancers adjacent to the promoter of the NR4A3 gene or the closely related NR4A2 gene, resulting in their aberrant overexpression. Transcriptome analyses revealed several distinct subgroups of AcCC tumors, including a subgroup that overexpressed both NR4A3 and MSANTD3. A poor survival subset of the tumors with high-grade transformation expressed NR4A3 and POMC as well as MYB, an oncogene that is the major driver in a different type of salivary gland tumor, adenoid cystic carcinoma. The combination of NR4A3 and MYB showed cooperativity in regulating a distinct set of genes. In addition, the ligand binding domain of NR4A3 directly bound the Myb DNA binding domain. Transformation assays indicated that, while overexpressed NR4A3 was sufficient to generate transformed colonies, the combination of NR4A3 plus Myb was more potent, leading to anchorage-independent growth and increased cellular invasiveness. The results confirm that NR4A3 and NR4A2 are the main driver genes of AcCC and suggest that concurrent overexpression of NR4A3 and MYB defines a subset of AcCC patients with high-grade transformation that display exceptionally poor outcome.

The effect of NR4A3-rs12686676 and XBP1-rs2269577 polymorphisms on type 2 diabetes mellitus susceptibility in an Iranian population: Case-control study

Many genes with different impacts on β-cell function, have been known as Type 2 diabetes mellitus (T2DM) risk genes. The purpose of this investigation was the evaluation of the potential correlation between rs12686676 and rs2269577 common polymorphisms of NR4A3 and XBP1 genes which are linked to β-cell function, and risk of T2DM in an Iranian population. In the present case-control investigation, 350 people affected by T2DM and 350 subjects in control group genotyped for NR4A3-rs12686676 by Tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method and XBP1-rs2269577 polymorphisms by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. According to results, no correlation between T2DM risk and NR4A3-rs12686676 polymorphism was found. However, we observed that CG genotype of XBP1-rs2269577 polymorphism could significantly decrease T2DM risk (OR = 0.6, 95%CI: 0.40–0.89, P = 0.01). Moreover, we detected that XBP1-rs2269577 SNP was related to the decreased risk of T2DM in dominant genetic model (OR = 0.5, 95%CI: 0.40–0.88, P = 0.01). In conclusion, there was no connection among pathogenesis of T2DM risk and NR4A3-rs12686676 polymorphism, however we found that heterozygote genotype (CG) of XBP1-rs2269577 SNP could decrease risk of T2DM in an Iranian population. Our result is the first report to show the protective role of rs2269577 SNP on diabetes risk.

Neonatal Tactile Stimulation Alters Behaviors in Heterozygous Serotonin Transporter Male Rats: Role of the Amygdala.

The serotonin transporter (SERT) gene, especially the short allele of the human serotonin transporter linked polymorphic region (5-HTTLPR), has been associated with the development of stress-related neuropsychiatric disorders. In line, exposure to early life stress in SERT knockout animals contributes to anxiety- and depression-like behavior. However, there is a lack of investigation of how early-life exposure to beneficial stimuli, such as tactile stimulation (TS), affects later life behavior in these animals. In this study, we investigated the effect of TS on social, anxiety, and anhedonic behavior in heterozygous SERT knockouts rats and wild-type controls and its impact on gene expression in the basolateral amygdala. Heterozygous SERT+/– rats were submitted to TS during postnatal days 8–14, for 10 min per day. In adulthood, rats were assessed for social and affective behavior. Besides, brain-derived neurotrophic factor (Bdnf) gene expression and its isoforms, components of glutamatergic and GABAergic systems as well as glucocorticoid-responsive genes were measured in the basolateral amygdala. We found that exposure to neonatal TS improved social and affective behavior in SERT+/– animals compared to naïve SERT+/– animals and was normalized to the level of naïve SERT+/+ animals. At the molecular level, we observed that TS per se affected Bdnf, the glucocorticoid-responsive genes Nr4a1, Gadd45β, the co-chaperone Fkbp5 as well as glutamatergic and GABAergic gene expression markers including the enzyme Gad67, the vesicular GABA transporter, and the vesicular glutamate transporter genes. Our results suggest that exposure of SERT+/– rats to neonatal TS can normalize their phenotype in adulthood and that TS per se alters the expression of plasticity and stress-related genes in the basolateral amygdala. These findings demonstrate the potential effect of a supportive stimulus in SERT rodents, which are more susceptible to develop psychiatric disorders.

De novo variants of NR4A2 are associated with neurodevelopmental disorder and epilepsy

PurposeThis study characterizes the clinical and genetic features of nine unrelated patients with de novo variants in the NR4A2 gene. MethodsVariants were identified and de novo origins were confirmed through trio exome sequencing in all but one patient. Targeted RNA sequencing was performed for one variant to confirm its splicing effect. Independent discoveries were shared through GeneMatcher. ResultsMissense and loss-of-function variants in NR4A2 were identified in patients from eight unrelated families. One patient carried a larger deletion including adjacent genes. The cases presented with developmental delay, hypotonia (six cases), and epilepsy (six cases). De novo status was confirmed for eight patients. One variant was demonstrated to affect splicing and result in expression of abnormal transcripts likely subject to nonsense-mediated decay. ConclusionOur study underscores the importance of NR4A2 as a disease gene for neurodevelopmental disorders and epilepsy. The identified variants are likely causative of the seizures and additional developmental phenotypes in these patients.

Identification and functional analysis of fourteen NR5A1 variants in patients with the 46 XY disorders of sex development

Human sex determination and differentiation is a complex process, during which NR5A1 plays a central role via the transcriptional regulation of key modulators involved in steroidogenesis. Approximately 8-15% of 46,XY DSD are caused by variants in the NR5A1 gene. Therefore, screening for variants in the NR5A1 gene was performed in a Chinese cohort of sixty-two 46,XY DSD patients with no AR or SRD5A2 variants via next-generation sequencing (NGS). Fourteen variants in the NR5A1 gene were identified in 16 patients from 14 unrelated families, including nine novel variants. These variants included eight heterozygote missense variants, two heterozygote frameshift variants, two heterozygote nonsense variants, one heterozygote nonframeshift deletion-insertion variant, and one homozygous missense variant. Functional assays showed that the transcriptional activity of the 11 variants was significantly reduced. In this study, 11 NR5A1 pathogenic variants were identified. These novel variants further expand the existing spectrum of the NR5A1 variants associated with 46,XY DSD, which will, in turn, assist in the molecular diagnosis of DSD.

An exome-wide exploration of cases of primary ovarian insufficiency uncovers novel sequence variants and candidate genes.

Primary ovarian insufficiency (POI) implies the cessation of menstruation for several months in women before the age of 40 years and is a major cause of infertility. The study of the contribution of genetic factors to POI has been fueled by the use of whole exome sequencing (WES). Here, to uncover novel causative pathogenic variants and risk alleles, WES has been performed in 12 patients with familial POI (eight unrelated index cases and two pairs of sisters) and six women with early menopause and family history of POI (four index cases and one pair of sisters). Likely causative variants in NR5A1 and MCM9 genes were identified as well as a variant in INHA that requires further investigation. Moreover, we have identified more than one candidate variant in 3 out of 15 familial cases. Taken together, our results highlight the genetic heterogeneity of POI and early menopause and support the hypothesis of an oligogenic inheritance of such conditions, in addition to monogenic inheritance.

Next-generation sequencing: toward an increase in the diagnostic yield in patients with apparently idiopathic spermatogenic failure.

A large proportion of patients with idiopathic spermatogenic failure (SPGF; oligozoospermia or nonobstructive azoospermia [NOA]) do not receive a diagnosis despite an extensive diagnostic workup. Recent evidence has shown that the etiology remains undefined in up to 75% of these patients. A number of genes involved in germ-cell proliferation, spermatocyte meiotic divisions, and spermatid development have been called into play in the pathogenesis of idiopathic oligozoospermia or NOA. However, this evidence mainly comes from case reports. Therefore, this study was undertaken to identify the molecular causes of SPGF. To accomplish this, 15 genes (USP9Y, NR5A1, KLHL10, ZMYND15, PLK4, TEX15, TEX11, MEIOB, SOHLH1, HSF2, SYCP3, TAF4B, NANOS1, SYCE1, and RHOXF2) involved in idiopathic SPGF were simultaneously analyzed in a cohort of 25 patients with idiopathic oligozoospermia or NOA, accurately selected after a thorough diagnostic workup. After next-generation sequencing (NGS) analysis, we identified the presence of rare variants in the NR5A1 and TEX11 genes with a pathogenic role in 3/25 (12.0%) patients. Seventeen other different variants were identified, and among them, 13 have never been reported before. Eleven out of 17 variants were likely pathogenic and deserve functional or segregation studies. The genes most frequently mutated were MEIOB, followed by USP9Y, KLHL10, NR5A1, and SOHLH1. No alterations were found in the SYCP3, TAF4B, NANOS1, SYCE1, or RHOXF2 genes. In conclusion, NGS technology, by screening a specific custom-made panel of genes, could help increase the diagnostic rate in patients with idiopathic oligozoospermia or NOA.

Cross‑validation of genes potentially associated with neoadjuvant chemotherapy and platinum‑based chemoresistance in epithelial ovarian carcinoma.

Ovarian carcinomas have the poorest prognosis and the highest mortality among gynecological malignancies. Neoadjuvant chemotherapy (NACT) is considered as a novel therapeutic strategy and an alternative treatment for advanced epithelial ovarian cancer (AEOC). The aim of the present study was to identify the core genes related to platinum-based NACT resistance in AEOC and to allow screening at the molecular level for the most appropriate ovarian cancer patients for NACT. We obtained three drug-resistant microarrays GSE114206, GSE41499 and GSE33482 from the Gene Expression Omnibus (GEO) database as well as a microarray representing NACT, GSE109934. Bioinformatics analysis revealed the nature of the four potential candidate genes for using in functional enrichment analyses and interaction network construction. The potential associations and possible genetic alterations among the DEGs were summarized using the STRING database in Cytoscape and the cBioPortal visualization tool, respectively. A total of 63 genes were identified as DEGs from GSE109934 representing NACT. From the drug-resistant GSE114206 and GSE41499 datasets, 106 DEGs containing 36 upregulated genes and 70 downregulated genes were selected, and from the drug-resistant GSE114206 and GSE33482 datasets, 406 DEGs with 157 upregulated genes and 249 downregulated genes were selected. The 36 upregulated DEGs and the 70 downregulated genes were notably abundant in the different categories. In KEGG pathway analysis, the 157 upregulated genes and the 249 downregulated genes were concentrated in distinctive signaling pathways. Four potential genes associated with NACT and platinum-based chemoresistance were screened, including nuclear factor of activated T-cells, cytoplasmic 1 (NAFTc1), Kruppel-like factor 4 (KLF4), nuclear receptor subfamily 4 group A member 3 (NR4A3) and hepatocyte growth factor (HGF). Our study showed that the mRNA expression levels of NAFTc1, NR4A3 and HGF were increased in drug-resistant OC cell lines (all P<0.01), whereas the mRNA expression levels of KLF4 were notably lower in the SKOV3-CDDP and HeyA8-CDDP cell line (all P<0.01) but higher in the A2780-CBP cell line. The NAFTc1, KLF4, NR4A3 and HGF genes may be potential therapeutic targets for NACT and platinum-based chemoresistance factors as well as candidate biomarkers in AEOC. Determination of the expression levels of these four genes in tumor tissues before planning NACT treatment or initial surgery would be beneficial for AEOC patients.

In This Issue

In This Issue

NR5A1 Gene Variants: Variable Phenotypes, New Variants, Different Outcomes

NR5A1 (nuclear receptor subfamily 5 group A member 1) is a transcriptional regulator of adrenal and gonadal development and function. Heterozygous and homozygous NR5A1 mutations have been described in people with 46,XY disorders of sex development (DSD). The clinical, endocrine, and genetic features of four 46,XY subjects with NR5A1 genetic variants (2 sisters, 2 boys) from 3 unrelated families are reported. All subjects presented with hypergonadotropic hypogonadism and abnormal pubertal progression. Markers of Sertoli cell function were more affected than those of Leydig cell function. Genetic investigation demonstrated the presence of different heterozygous NR5A1 genetic variants. In the boys, pathogenetic NR5A1 gene variants were found that had been previously reported. The 2 sisters carried a new genetic variant in exon 4, and in silico analysis and ACMG classification indicated its pathogenicity. The data confirmed that NR5A1 gene mutations may present with variable genital phenotypes. Anyway, reproductive function was always impaired. Any clinical or endocrine data seem to be unable to differentiate these patients from other 46,XY DSD cases, suggesting that molecular analysis must be warranted. In subjects with NR5A1 mutations, different decisions in sex assignment may permit satisfying somatic and psychological outcome, but any option requires hormonal substitutive therapy from adolescence onward.

In search of markers useful for evaluation of graft patency - molecular analysis of ‘muscle system process’ for internal thoracic artery and saphenous vein conduits

Abstract Coronary artery bypass graft (CABG) is the surgical method most commonly used to treat coronary artery disease (CAD). The vessels that are used in CABG are usually the internal thoracic artery (ITA) and the saphenous vein (SV). Transplant patency is one of the most important factors affecting transplant success. In this study, we used an expressive microarray method, approved by RT-qPCR, for transcriptome analysis of arterial and venous grafts. In the search for potential molecular factors, we analyzed gene ontologies of different expression based on the muscular system. Among interesting groups, we distinguished muscle cell proliferation, muscle contraction, muscle system process, regulation of smooth muscle cell proliferation and smooth muscle cell proliferation. The highest increase in gene expression was observed in: ACTN2, RBPMS2, NR4A3, KCNA5, while the smallest decrease in expression was shown by the P2RX1, KCNH2, DES and MYOT genes. Particularly noteworthy are the ACTN2 and NR4A3 genes, which can have a significant impact on vascular patency. ACTN2 is a gene that can affect the formation of atherosclerotic plaques, while NR4A3 occurs in 4 of the 5 ontological groups discussed and can affect the inflammatory process in the blood vessel. To summarize, the presented study provided valuable insight into the molecular aspects characterizing the vessels used in CABG, and in particular identified genes that may be the target for further studies on duct patency. Running title: CABG grafts’ molecular analysis of ‘muscle system process’

A novel voluntary weightlifting model in mice promotes muscle adaptation and insulin sensitivity with simultaneous enhancement of autophagy and mTOR pathway.

Our understanding of the molecular mechanisms underlying adaptations to resistance exercise remains elusive despite the significant biological and clinical relevance. We developed a novel voluntary mouse weightlifting model, which elicits squat-like activities against adjustable load during feeding, to investigate the resistance exercise-induced contractile and metabolic adaptations. RNAseq analysis revealed that a single bout of weightlifting induced significant transcriptome responses of genes that function in posttranslational modification, metabolism, and muscle differentiation in recruited skeletal muscles, which were confirmed by increased expression of fibroblast growth factor-inducible 14 (Fn14), Down syndrome critical region 1 (Dscr1) and Nuclear receptor subfamily 4, group A, member 3 (Nr4a3) genes. Long-term (8 weeks) voluntary weightlifting training resulted in significantly increases of muscle mass, protein synthesis (puromycin incorporation in SUnSET assay) and mTOR pathway protein expression (raptor, 4e-bp-1, and p70S6K proteins) along with enhanced muscle power (specific torque and contraction speed), but not endurance capacity, mitochondrial biogenesis, and fiber type transformation. Importantly, weightlifting training profound improved whole-body glucose clearance and skeletal muscle insulin sensitivity along with enhanced autophagy (increased LC3 and LC3-II/I ratio, and decreased p62/Sqstm1). These data suggest that resistance training in mice promotes muscle adaptation and insulin sensitivity with simultaneous enhancement of autophagy and mTOR pathway.

The Regulation of Intestinal Smooth Muscle Cell Proliferation in Crohn’s Disease – Is NR4A1 a Novel Target to Treat Fibrostenosis?

Intestinal fibrosis and stricture formation are common complications of Crohn’s disease (CD). Recently, the stricturing phenotype has been recognized to be primarily attributable to hypertrophy/hyperplasia of smooth muscle, rather than an increase in fibrosis alone. Despite advances in treatment of CD, current therapies do little to prevent or reverse strictures. NR4A1 is an orphan nuclear receptor that has been reported as anti‐fibrotic in non‐intestinal systems and exhibits anti‐proliferative effects in smooth muscle cells (SMCs). NR4A1 gene variants have been associated with increased risk of inflammatory bowel disease, however, the mechanisms regulating NR4A1 expression and its role in intestinal SMC function have not been investigated.We sought to characterize and understand the role of NR4A1 activation in the regulation of mitogen‐induced proliferation of intestinal SMCs. We hypothesized that the activation of NR4A1 negatively regulates proliferative signaling and cell survival in intestinal SMCs.To test the hypothesis, primary intestinal SMCs were isolated from Nr4a1+/+ and Nr4a1−/− mice. Furthermore, a commercially sourced human primary intestinal SMC line was used. To assess the response of SMCs to culture and identify growth differences, proliferation was measured via trypan blue exclusion and EdU incorporation. In mouse and human SMCs, proliferation was induced by platelet‐derived growth factor‐BB (PDGF‐BB) and NR4A1 activation was assessed by pretreating with selective agonists, cytosporone‐B (Csn‐B) and 6‐mercaptopurine (6‐MP), at various concentrations. Mass spectrometry was used to characterize proteomic differences between Nr4a1+/+ and Nr4a1−/− SMCs. Expression levels of NR4A1 were assessed by qPCR and western blot after mitogen exposure and Csn‐B treatment.Nr4a1−/− cells exhibited a significantly higher rate of proliferation compared to Nr4a1+/+ cells, under both basal and mitogen‐exposed conditions. Proteomic analysis showed that Nr4a1−/− SMCs exhibited increased expression of proteins related to the cell cycle and metabolism, compared to Nr4a1+/+ SMCs. Pretreating human intestinal SMCs with Csn‐B and 6‐MP significantly attenuated proliferation induced by PDGF‐BB. Similar effects were observed in Nr4a1+/+ SMCs, however, the anti‐proliferative effect of Csn‐B was absent in Nr4a1−/− cells. Furthermore, NR4A1 expression was rapidly induced by Csn‐B and PDGF‐BB, the latter response suggesting the existence of a potential negative feedback mechanism to control mitogen‐induced SMC proliferation.Our results suggest that NR4A1 is a critical regulator of intestinal SMC proliferation and its induction by mitogens may contribute to a negative feedback loop to control smooth muscle growth. These data support targeting NR4A1 to treat excessive smooth muscle hypertrophy/hyperplasia that contributes to tissue remodelling observed in fibrostenotic CD.Support or Funding InformationCanadian Institutes of Health Research

The Transcription Factor NR4A2 Plays an Essential Role in Driving Prolactin Expression in Female Pituitary Lactotropes.

Differentiation of the hormone-producing cells of the pituitary represents an informative model of cell fate determination. The generation and maintenance of 2 pituitary lineages, the growth hormone (GH)- producing somatotropes and the prolactin (PRL)- producing lactotropes, are dependent on the pituitary-specific transcription factor, POU1F1. While POU1F1 is expressed in both cell types, and plays a role in activation of both the Gh and Prl genes, expression of Gh and Prl is restricted to somatotropes and lactotropes, respectively. These observations imply the existence of additional factors that contribute to the somatotrope and lactotrope identities and their hormone expressions. Prior transcriptome analysis of primary somatotropes and lactotropes isolated from the mouse pituitary identified enrichment of a transcription factor, Nr4a2, in the lactotropes. Nr4a2 was shown in a cell culture model to bind the Prl promoter at a position adjacent to Pou1f1 and to synergize with Pou1f1 in driving Prl transcription. Here we demonstrate in vivo the role of Nr4a2 as an enhancer of Prl expression by conditional gene inactivation of the Nr4a2 gene in mouse lactotropes. We demonstrate that nuclear orphan receptor transcription factor (NR4A2) binding at the Prl promoter is dependent on actions of POU1F1; while POU1F1 is essential to loading polymerase (Pol) II on the Prl promoter, Nr4a2 plays a role in enhancing Pol II release into the Prl gene body. These studies establish an in vivo role of Nr4a2 in enhancing Prl expression in mouse lactotropes, explore its mechanism of action, and establish a system for further study of the lactotrope lineage in the pituitary.