scholarly journals Coactivation of SF-1-Mediated Transcription of Steroidogenic Enzymes by Ubc9 and PIAS1

Endocrinology ◽  
2011 ◽  
Vol 152 (6) ◽  
pp. 2266-2277 ◽  
Author(s):  
Noriko Suda ◽  
Hirotaka Shibata ◽  
Isao Kurihara ◽  
Yayoi Ikeda ◽  
Sakiko Kobayashi ◽  
...  
Keyword(s):  
Adrenal Cortex ◽  
Gene Promoter ◽  
Orphan Receptor ◽  
Mrna Levels ◽  
Steroidogenic Enzyme ◽  
Steroidogenic Factor 1 ◽  
Cyp17 Gene ◽  
Nuclear Orphan Receptor ◽  
Interfering Rna

Steroidogenic factor-1 (SF-1) is a nuclear orphan receptor, which is essential for adrenal development and regulation of steroidogenic enzyme expression. SF-1 is posttranslationally modified by small ubiquitin-related modifier-1 (SUMO-1), thus mostly resulting in attenuation of transcription. We investigated the role of sumoylation enzymes, Ubc9 and protein inhibitors of activated STAT1 (PIAS1), in SF-1-mediated transcription of steroidogenic enzyme genes in the adrenal cortex. Coimmunoprecipitation assays showed that both Ubc9 and PIAS1 interacted with SF-1. Transient transfection assays in adrenocortical H295R cells showed Ubc9 and PIAS1 potentiated SF-1-mediated transactivation of reporter constructs containing human CYP17, CYP11A1, and CYP11B1 but not CYP11B2 promoters. Reduction of endogenous Ubc9 and PIAS1 by introducing corresponding small interfering RNA significantly reduced endogenous CYP17, CYP11A1, and CYP11B1 mRNA levels, indicating that they normally function as coactivators of SF-1. Wild type and sumoylation-inactive mutants of Ubc9 and PIAS1 can similarly enhance the SF-1-mediated transactivation of the CYP17 gene, indicating that the coactivation potency of Ubc9 and PIAS1 is independent of sumoylation activity. Chromatin immunoprecipitation assays demonstrated that SF-1, Ubc9, and PIAS1 were recruited to an endogenous CYP17 gene promoter in the context of chromatin in vivo. Immunohistochemistry and Western blotting showed that SF-1, Ubc9, and PIAS1 were expressed in the nuclei of the human adrenal cortex. In cortisol-producing adenomas, the expression pattern of SF-1 and Ubc9 were markedly increased, whereas that of PIAS1 was decreased compared with adjacent normal adrenals. These results showed the physiological roles of Ubc9 and PIAS1 as SF-1 coactivators beyond sumoylation enzymes in adrenocortical steroidogenesis and suggested their possible pathophysiological roles in human cortisol-producing adenomas.

scholarly journals DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

2013 ◽  
Vol 451 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Claudia C. S. Chini ◽  
Carlos Escande ◽  
Veronica Nin ◽  
Eduardo N. Chini
Keyword(s):  
Breast Cancer ◽  
Nuclear Receptor ◽  
Clock Genes ◽  
Mrna Levels ◽  
Protein Levels ◽  
The Stability ◽  
And Function ◽  
Interfering Rna ◽  
In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

The nuclear orphan receptors COUP-TFII and Ear-2 act as silencers of the human oxytocin gene promoter

1997 ◽  
Vol 19 (2) ◽  
pp. 163-172 ◽  
Author(s):  
K Chu ◽  
HH Zingg
Keyword(s):  
Receptor Binding ◽  
Promoter Activity ◽  
Transcriptional Activity ◽  
Gene Promoter ◽  
Fine Tuning ◽  
Orphan Receptor ◽  
Expression Vectors ◽  
Mobility Shift ◽  
Interaction Sites

We have previously shown that COUP-TFII and Ear-2, two members of the nuclear orphan receptor family, are able to repress oestrogen-stimulated transcriptional activity of the human oxytocin (OT) gene promoter by binding to a site that overlaps with the oestrogen response element (ERE) present in the 5' flanking region of the gene. Although most nuclear receptor-mediated transcriptional repression conforms with the paradigm of passive repression and involves competitive binding to an activator site, active repression, i.e. silencing of basal promoter activity, has been observed in a limited number of cases. Here we show by co-transfection experiments using COUP-TFII and Ear-2 expression vectors and reporter constructs containing OT gene promoter fragments linked to the chloramphenicol acetyltransferase gene that both COUP-TFII and Ear-2 are capable of silencing basal OT gene promoter activity by 54 and 75% respectively. 5' Deletion and footprint analyses revealed two areas of functionally important interaction sites: (1) a direct TGACC(T/C) repeat overlapping the ERE and (2) a more promoter-proximal area centred at - 90 containing three imperfect direct repeats (R1-R3) spaced by four nucleotides each. Mutagenesis of reporter constructs as well as electrophoretic mobility-shift assays demonstrated that each of the three proximal repeats R1-R3 contributed to orphan receptor binding and the silencing effect. Inasmuch as the orphan receptor-binding sites are not involved in mediating basal transcriptional activity of the OT gene promoter, the observed effects are best interpreted as active repression or promoter silencing. Moreover, since COUP-TFII and Ear-2 are both co-expressed in OT-expressing uterine epithelial cells, the novel transcriptional effects described here are likely to be of functional importance in the fine-tuning of uterine OT gene expression in vivo.

Selective inhibition of sphingosine kinase-1 protects adipose tissue against LPS-induced inflammatory response in Zucker diabetic fatty rats

2014 ◽  
Vol 307 (5) ◽  
pp. E437-E446 ◽  
Author(s):  
Monica Tous ◽  
Raquel Ferrer-Lorente ◽  
Lina Badimon
Keyword(s):  
Adipose Tissue ◽  
Inflammatory Response ◽  
Sphingosine Kinase ◽  
Selective Inhibition ◽  
Mrna Levels ◽  
Sphingosine Kinase 1 ◽  
Zucker Diabetic Fatty Rats ◽  
Cc Chemokines ◽  
Interfering Rna

Obesity is associated with a state of chronic inflammation. The chemokine (C-C motif) ligand 5 (CCL5) has been proposed to modulate the inflammatory response in adipose tissue (AT). However, the mechanisms underlying CCL5 upregulation in AT remain undefined. The objective of the present study was to evaluate whether the enzyme sphingosine kinase-1 (SK1) would modulate the expression of CCL5 and other inflammatory biomarkers in primary adipocytes and its potential role in lipopolysaccharide (LPS)-induced AT inflammation in a rat model of diabetes. To address this, LPS-stimulated primary adipocytes and 3T3-L1 cells were treated with a SK inhibitor, and the expression of Ccl5 and other CC chemokines were studied. Moreover, the effect of SK1 knockdown on cytokine production was analyzed in 3T3-L1 cells by transfection of SK1-specific small-interfering RNA (siRNA). The anti-inflammatory effects of SK inhibitor in AT were also investigated in vivo using the Zucker lean normoglycemic control (ZLC) rats. LPS treatment stimulated Ccl5, IL-6, pentraxin 3 ( Ptx3), and Tnfα mRNA expression in primary adipocytes and 3T3-L1 cells, whereas pharmacologically and siRNA-mediated SK1 inhibition strongly reduced mRNA levels of proinflammatory cytokines in these cells. Similarly, administration of SK inhibitor to ZLC rats prevented the LPS-induced inflammatory response in AT. Our data demonstrate a role for SK1 in endotoxin-induced cytokine expression in adipocytes and suggest that inhibition of SK1 may be a potential therapeutic tool in the prevention and treatment of chronic and common metabolic disorders, including obesity, insulin-resistance, and type 2 diabetes.

Nur77 and Nurr1, Two Novel Players In MSC Migration and Immune Function.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3854-3854 ◽  
Author(s):  
Marijke Maijenburg ◽  
Christian Gilissen ◽  
Joris Veltman ◽  
Marion Kleijer ◽  
Cris Mudde-Jadra ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Pore Size ◽  
Cytokine Production ◽  
Immunomodulatory Effect ◽  
Orphan Receptor ◽  
Mrna Levels ◽  
Migratory Capacity ◽  
Nuclear Orphan Receptor ◽  
Receptor Family

Abstract Abstract 3854 Mesenchymal stromal cells (MSC) have a promising potential for cellular therapies, based on their immunomodulatory functions and their differentiation capacity. However, it remains unclear how MSC exert their functions and especially homing towards effector sites is poorly understood. An imperative for future therapies is to understand the process of MSC migration. We have previously shown that only 10–20% of culture-expanded MSC is capable to migrate in vitro, but these migratory cells can not be discriminated by markers. To identify genes specific for migratory MSC, we performed a gene expression study. Using a Transwell system (12mm pore size), migrating and non-migrating fetal bone marrow MSC (FBMSC) were physically separated and RNA was extracted. MSC that were exposed to a SDF-1 gradient (0.4mm pore size) and cultured FBMSC were included as controls. Only 9 genes were differentially expressed between migratory and non-migratory MSC. This set did not include cell surface markers that enable selection for a migratory subset. The nuclear orphan receptor family members Nurr1 and Nur77 were 2-fold upregulated in migratory MSC. These results were confirmed by RQ-PCR. Nurr1 and Nur77 are members of the NR4A nuclear orphan receptor family and were first described as early response transcription factors upon growth factor stimulation. We observed that the chemokine SDF-1 and the growth factor PDGF-BB increased the expression of Nur77 and Nurr1 in MSC. To investigate the role of Nur77 and Nurr1 in migration, FBMSC were transduced with lentivirus to achieve overexpression. Both overexpression of Nur77 and Nurr1 increased specific migration towards SDF-1 compared to Mock transduced FBMSC (n=4) (Nur77 mean 297% ± 186 relative to Mock, p≤ 0.014; Nurr1 mean 227% ± 79, p≤ 0.014). We previously described that cell cycle influences MSC migration, with S- and G2/M-phase of the cell cycle negatively influencing migration. In MSC cultures overexpressing Nur77 or Nurr1, the percentage of cells in S-phase of the cell cyle was significantly reduced (Nur77 15.2±7.8%, p≤0.007; Nurr1 16.4±9.1%, p≤0.039) compared to Mock-transduced cells (24.0±8.9%). Possibly, part of the Nur77 and Nurr1 effect is regulated by an effect on cell cycle. Together these data suggest that Nur77 and Nurr1 are involved in migration of MSC, which offers perspectives to modulate migration of MSC. Because these nuclear orphan receptors were not implied in MSC biology to date, we study their immunomodulatory effect. NR4A genes modulate immune responses in a variety of cells by influencing cytokine production. The immunomodulatory effect of MSC also seems to be based on secretion of a wide variety of cytokines, chemokines and growth factors. We therefore investigated whether Nur77 and Nurr1 play a role in cytokine and growth factor production in MSC. Compared to Mock transduced MSC, overexpression of Nur77 and Nurr1 in MSC resulted in increased basal levels of IL-6 protein (Nur77 3.4±2.4 fold, p≤0.007; Nurr1 3.8±3.1 ns) and IL-8 protein (Nur77 5.9±3.3 fold, p≤0.046; Nurr1 4.7±2.2, p≤0.031) and HGF mRNA expression (Nur77 5.4±0.8 fold, p≤0.021; Nurr1 3.4±1.18, p≤0.026). This was further enhanced upon stimulation with TNFα for 24hrs relative to the unstimulated Mock (IL-6 Mock 22.9±19.5 fold; Nur77 35.1±21.9 fold; Nurr1 35.8±24.1 fold; IL-8 Mock 58±37 fold; Nur77 160±141 fold; Nurr1 262±181 fold). HGF mRNA levels were slightly elevated by stimulation with IFNγ for 24hrs (Mock 1.8±1.6 fold; Nur77 7.0±3.8 fold; Nurr1 7.1±1.7 fold; ns). Expression levels of IDO1 and TGF-ß, also involved in immune modulation by MSC, were not influenced by Nur77 or Nurr1. In conclusion, we identified two novel genes involved in MSC migration. Intriguingly these genes are also involved in cytokine production, suggesting that the fraction with the best migratory capacity is able to modulate the immune response at its effector site distinct from the bulk of culture expanded MSC that lack migratory capacity. Modulation of Nur77 and Nurr1 expression in MSC could therefore offer perspectives to ameliorate cellular therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Foxa1 and Foxa2 Regulate α-Cell Differentiation, Glucagon Biosynthesis, and Secretion

Endocrinology ◽  
2014 ◽  
Vol 155 (10) ◽  
pp. 3781-3792 ◽  
Author(s):  
Mounia Heddad Masson ◽  
Caroline Poisson ◽  
Audrey Guérardel ◽  
Aline Mamin ◽  
Jacques Philippe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
Target Genes ◽  
Adult Mouse ◽  
Glucagon Secretion ◽  
Mrna Levels ◽  
Forkhead Box ◽  
Interfering Rna

Abstract The Forkhead box A transcription factors are major regulators of glucose homeostasis. They show both distinct and redundant roles during pancreas development and in adult mouse β-cells. In vivo ablation studies have revealed critical implications of Foxa1 on glucagon biosynthesis and requirement of Foxa2 in α-cell terminal differentiation. In order to examine the respective role of these factors in mature α-cells, we used small interfering RNA (siRNA) directed against Foxa1 and Foxa2 in rat primary pancreatic α-cells and rodent α-cell lines leading to marked decreases in Foxa1 and Foxa2 mRNA levels and proteins. Both Foxa1 and Foxa2 control glucagon gene expression specifically through the G2 element. Although we found that Foxa2 controls the expression of the glucagon, MafB, Pou3f4, Pcsk2, Nkx2.2, Kir6.2, and Sur1 genes, Foxa1 only regulates glucagon gene expression. Interestingly, the Isl1 and Gipr genes were not controlled by either Foxa1 or Foxa2 alone but by their combination. Foxa1 and Foxa2 directly activate and bind the promoter region the Nkx2.2, Kir6.2 and Sur1, Gipr, Isl1, and Pou3f4 genes. We also demonstrated that glucagon secretion is affected by the combined effects of Foxa1 and Foxa2 but not by either one alone. Our results indicate that Foxa1 and Foxa2 control glucagon biosynthesis and secretion as well as α-cell differentiation with both common and unique target genes.

scholarly journals A Unique Pharmacophore for Activation of the Nuclear Orphan Receptor Nur77 In vivo and In vitro

2010 ◽  
Vol 70 (9) ◽  
pp. 3628-3637 ◽  
Author(s):  
Jing-jing Liu ◽  
Hui-ni Zeng ◽  
Lian-ru Zhang ◽  
Yan-yan Zhan ◽  
Yan Chen ◽  
...  
Keyword(s):  
Orphan Receptor ◽  
Nuclear Orphan Receptor

scholarly journals Transcriptional Regulation of HumanRev-erbαGene Expression by the Orphan Nuclear Receptor Retinoic Acid-related Orphan Receptor α

2002 ◽  
Vol 277 (51) ◽  
pp. 49275-49281 ◽  
Author(s):  
Eric Raspè ◽  
Gisèle Mautino ◽  
Caroline Duval ◽  
Coralie Fontaine ◽  
Hélène Duez ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Hepg2 Cells ◽  
Gene Promoter ◽  
Orphan Receptor ◽  
Similar Response ◽  
Orphan Nuclear Receptor ◽  
Orphan Nuclear Receptors ◽  
Mrna Accumulation

The Rev-erb and retinoic acid-related orphan receptors (ROR) are two related families of orphan nuclear receptors that recognize similar response elements but have opposite effects on transcription. Recently, theRev-erbαgene promoter has been characterized and shown to harbor a functional Rev-erbα-binding site known as Rev-DR2, responsible for negative feedback down-regulation of promoter activity by Rev-erbα itself. The present study aimed to investigate whetherRev-erbαgene expression is regulated by RORα. Gel shift analysis demonstrated thatin vitrotranslated hRORα1 protein binds to the Rev-DR2 site, both as monomer and dimer. Chromatin immunoprecipitation assays demonstrated that binding of RORα to this site also occurredin vivoin human hepatoma HepG2 cells. The Rev-DR2 site was further shown to be functional as it conferred hRORα1 responsiveness to a heterologous promoter and to the natural humanRev-erbαgene promoter in these cells. Mutation of this site in the context of the naturalRev-erbαgene promoter abolished its activation by RORα, indicating that this site plays a key role in hRORα1 action. Finally, adenoviral overexpression of hRORα1 in HepG2 cells led to enhanced hRev-erbα mRNA accumulation, further confirming the physiological importance of RORα1 in the regulation of Rev-erbα expression.

scholarly journals In vivo oestrogenic modulation of Egr1 and Pitx1 gene expression in female rat pituitary gland

2014 ◽  
Vol 53 (3) ◽  
pp. 355-366 ◽  
Author(s):  
Alina Gajewska ◽  
Andrzej P Herman ◽  
Ewa Wolińska-Witort ◽  
Kazimierz Kochman ◽  
Lech Zwierzchowski
Keyword(s):  
Pituitary Gland ◽  
Mrna Expression ◽  
Gnrh Agonist ◽  
Gene Promoter ◽  
Specific Response ◽  
Mrna Levels ◽  
Female Rat ◽  
Promoter Activation ◽  
Rat Pituitary

EGR1 and PITX1 are transcription factors required for gonadotroph cell Lhb promoter activation. To determine changes in Egr1 and Pitx1 mRNA levels in central and peripheral pituitary stimulations, an in vivo model based on i.c.v. pulsatile (1 pulse/0.5 h over 2 h) GnRH agonist (1.5 nM buserelin) or antagonist (2 nM antide) microinjections was used. The microinjections were given to ovariectomised and 17β-oestradiol (E2) (3×20 μg), ERA (ESR1) agonist propyl pyrazole triol (PPT) (3×0.5 mg), ERB (ESR2) agonist diarylpropionitrile (DPN) (3×0.5 mg) s.c. pre-treated rats 30 min after last pulse anterior pituitaries were excised. Relative mRNA expression was determined by quantitative RT-PCR (qRT-PCR). Results revealed a gene-specific response for GnRH and/or oestrogenic stimulations in vivo. Buserelin pulses enhanced Egr1 expression by 66% in ovariectomised rats, whereas the oestradiol-supplemented+i.c.v. NaCl-microinjected group showed a 50% increase in Egr1 mRNA expression. The oestrogenic signal was transmitted via ERA (ESR1) and ERB (ESR2) activation as administration of PPT and DPN resulted in 97 and 62%, respectively, elevation in Egr1 mRNA expression. A synergistic action of GnRH agonist and 17β-oestradiol (E2) stimulation of the Egr1 gene transcription in vivo were found. GnRHR activity did not affect Pitx1 mRNA expression; regardless of NaCl, buserelin or antide i.c.v. pulses, s.c. oestrogenic supplementation (with E2, PPT or DPN) consistently decreased (by −46, −48 and −41% respectively) the Pitx1 mRNA in the anterior pituitary gland. Orchestrated Egr1 and Pitx1 activities depending on specific central and peripheral regulatory inputs could be responsible for physiologically variable Lhb gene promoter activation in vivo.

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

Endocrinology ◽  
2020 ◽  
Vol 161 (5) ◽  
Author(s):  
Michael T Peel ◽  
Yugong Ho ◽  
Stephen A Liebhaber
Keyword(s):  
Transcription Factor ◽  
Cell Fate ◽  
Cell Types ◽  
Orphan Receptor ◽  
Pol Ii ◽  
A Cell ◽  
Mouse Pituitary ◽  
Nuclear Orphan Receptor

Abstract 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.

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