Transcriptional and Metabolic Effects of Glucocorticoid Receptor α and β Signaling in Zebrafish

In humans and zebrafish, 2 glucocorticoid (GC) receptor (GR) splice variants exist: the canonical GR α-isoform (GRα), and the GRβ. In the present study, we have used the zebrafish model system in order to reveal genes affected by each of these 2 receptor isoforms. By injecting zebrafish embryos with different splice-blocking morpholinos, we could knock down both GR isoforms or could target the alternative splicing of the GR pre-mRNA in favor of the GRβ. In addition, specific GRβ overexpression was achieved by injecting mRNA. Embryos were treated with the synthetic GC dexamethasone, and transcriptome analysis was performed. Two distinct gene clusters were found that were regulated by GRα: one that was regulated by GRα under basal conditions (presence of endogenous cortisol only), and one that was regulated upon increased activation of GRα (using a pharmacological dose of dexamathasone). GRβ may act as a dominant-negative inhibitor of GRα when GRβ is overexpressed and the GRα expression level is knocked down simultaneously. However, without GRα knockdown, no evidence for this activity was found. In addition, the data indicate regulation of gene transcription through other mechanisms of action by GRβ. We also investigated the concentrations of several metabolites using nuclear magnetic resonance spectroscopy. We found that dexamethasone treatment and knockdown of GRα together with overexpression of GRβ had opposite effects on glucose, amino acid, and fatty acid levels. Thus, we have shed new light on the molecular mechanisms of GC-induced effects on metabolism, which are known to increase the risk of obesity, hyperglycemia, and diabetes.

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Discovery of a Functional Glucocorticoid Receptor β-Isoform in Zebrafish

Endocrinology ◽

10.1210/en.2007-1364 ◽

2007 ◽

Vol 149 (4) ◽

pp. 1591-1599 ◽

Cited By ~ 112

Author(s):

Marcel J. M. Schaaf ◽

Danielle Champagne ◽

Ivo H. C. van Laanen ◽

Diane C. W. A. van Wijk ◽

Annemarie H. Meijer ◽

...

Keyword(s):

Animal Model ◽

Glucocorticoid Receptor ◽

Developmental Stages ◽

Splice Variants ◽

The Body ◽

Dominant Negative ◽

Mrna Levels ◽

Receptor Isoforms ◽

Asthma Patients ◽

Dominant Negative Inhibitor

In humans, two glucocorticoid receptor (GR) splice variants exist: GRα and GRβ, which are identical between amino acids 1–727 and then diverge. Whereas GRα (the canonical GR) acts as a ligand-activated transcription factor, GRβ does not bind traditional glucocorticoid agonists, lacks GRα’s transactivational activity, and acts as a dominant-negative inhibitor of GRα. It has been suggested that this receptor isoform is involved in the induction of glucocorticoid resistance in asthma patients. Unfortunately, a GR β-isoform has been detected in only humans, and therefore, an animal model for studies on this isoform is lacking. In the present study, we demonstrate that in zebrafish a GR isoform exists that diverges from the canonical zebrafish GR at the same position as human GRβ from human GRα. The zebrafish GR β-isoform acts as a dominant-negative inhibitor in reporter assays, and the extent of inhibition and the effective GRα/GRβ ratio is similar to studies performed with the human GR isoforms. In addition, the subcellular localization of zebrafish GRβ is similar to its human equivalent. Finally, expression levels of GRα and GRβ were determined in adult zebrafish tissues and at several developmental stages. Both receptor isoforms were detected throughout the body, and GRβ mRNA levels were relatively low compared with GRα mRNA levels, as in humans. Thus, for the first time, a GR β-isoform has been identified in a nonhuman animal species, shedding new light on the relevance of this GR splice variant and providing a versatile animal model for studies on the GR system.

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Associations between promoter usage and alternative splicing of the glucocorticoid receptor gene

Journal of Molecular Endocrinology ◽

10.1677/jme.1.02117 ◽

2007 ◽

Vol 38 (1) ◽

pp. 91-98 ◽

Cited By ~ 42

Author(s):

Henk Russcher ◽

Virgil A S H Dalm ◽

Frank H de Jong ◽

Albert O Brinkmann ◽

Leo J Hofland ◽

...

Keyword(s):

Glucocorticoid Receptor ◽

Splice Variants ◽

Receptor Gene ◽

Dominant Negative ◽

Multiple Promoters ◽

Glucocorticoid Receptor Gene ◽

Dominant Negative Inhibitor ◽

Exon 1 ◽

Promoter Usage ◽

Different Tissues

The glucocorticoid receptor (GR) is widely expressed in various tissues throughout the human body. At least three different 3′-splice variants of the GR have been reported: GR-α, which is functionally active; GR-β, which is a dominant negative inhibitor of GR-α function; and GR-P, which is thought to activate the function of GR-α. At least seven different variants for exon 1 exist, 1A–1F and 1H, each with its own promoter. In this study, we explored if tissue-specific splicing of the 3′-end variants of the GR is influenced by alternative promoter usage. cDNAs of different tissues and cell lines were used to investigate which part of transcripts carrying each of the three major variants for exons 1, 1A, 1B, or 1C, encodes for the splice variants GR-α, GR-β, and GR-P. Our data demonstrate that the expression of GR-α is preferentially regulated by promoter 1C and that for the expression of GR-P promoter 1B is predominantly used. This indicates that regulation of GR splice variants could partly occur through selective use of the multiple promoters, and that this is another way to sensitize cells and tissues to the different activities of the GR isoforms.

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Human GH Receptor-IGF-1 Receptor Interaction: Implications for GH Signaling

Molecular Endocrinology ◽

10.1210/me.2014-1174 ◽

2014 ◽

Vol 28 (11) ◽

pp. 1841-1854 ◽

Cited By ~ 20

Author(s):

Yujun Gan ◽

Ashiya Buckels ◽

Ying Liu ◽

Yue Zhang ◽

Andrew J. Paterson ◽

...

Keyword(s):

Gene Expression ◽

Tyrosine Kinase ◽

Dominant Negative ◽

Janus Kinase ◽

Metabolic Effects ◽

Receptor Interaction ◽

Tyrosine Kinase Receptor ◽

Lncap Cells ◽

Gh Receptor ◽

Dominant Negative Inhibitor

GH signaling yields multiple anabolic and metabolic effects. GH binds the transmembrane GH receptor (GHR) to activate the intracellular GHR-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signals, including signal transducer and activator of transcription 5 (STAT5) activation and IGF-1 gene expression. Some GH effects are partly mediated by GH-induced IGF-1 via IGF-1 receptor (IGF-1R), a tyrosine kinase receptor. We previously demonstrated in non-human cells that GH causes formation of a GHR-JAK2-IGF-1R complex and that presence of IGF-1R (even without IGF-1 binding) augments proximal GH signaling. In this study, we use human LNCaP prostate cancer cells as a model system to further study the IGF-1R's role in GH signaling. GH promoted JAK2 and GHR tyrosine phosphorylation and STAT5 activation in LNCaP cells. By coimmunoprecipitation and a new split luciferase complementation assay, we find that GH augments GHR/IGF-1R complex formation, which is inhibited by a Fab of an antagonistic anti-GHR monoclonal antibody. Short hairpin RNA-mediated IGF-1R silencing in LNCaP cells reduced GH-induced GHR, JAK2, and STAT5 phosphorylation. Similarly, a soluble IGF-1R extracellular domain fragment (sol IGF-1R) interacts with GHR in response to GH and blunts GH signaling. Sol IGF-1R also markedly inhibits GH-induced IGF-1 gene expression in both LNCaP cells and mouse primary osteoblast cells. On the basis of these and other findings, we propose a model in which IGF-1R augments GH signaling by allowing a putative IGF-1R-associated molecule that regulates GH signaling to access the activated GHR/JAK2 complex and envision sol IGF-1R as a dominant-negative inhibitor of this IGF-1R-mediated augmentation. Physiological implications of this new model are discussed.

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Cryo–electron microscopy structure of the antidiuretic hormone arginine-vasopressin V2 receptor signaling complex

Science Advances ◽

10.1126/sciadv.abg5628 ◽

2021 ◽

Vol 7 (21) ◽

pp. eabg5628

Author(s):

Julien Bous ◽

Hélène Orcel ◽

Nicolas Floquet ◽

Cédric Leyrat ◽

Joséphine Lai-Kee-Him ◽

...

Keyword(s):

Electron Microscopy ◽

Antidiuretic Hormone ◽

Arginine Vasopressin ◽

Molecular Mechanisms ◽

Particle Analysis ◽

Resonance Spectroscopy ◽

Gpcr Signaling ◽

Signaling Complex ◽

Cryo Electron Microscopy ◽

V2 Receptor

The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the Gs protein, promoting kidney water reabsorption. Molecular mechanisms underlying activation of this critical G protein–coupled receptor (GPCR) signaling system are still unknown. To fill this gap of knowledge, we report here the cryo–electron microscopy structure of the AVP-V2R-Gs complex. Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and nuclear magnetic resonance spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes. They reveal an original receptor-Gs interface in which the Gαs subunit penetrates deep into the active V2R. The structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases. Our study provides important structural insights into the function of this clinically relevant GPCR signaling complex.

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Aerobic Exercise Induces Alternative Splicing of Neurexins in Frontal Cortex

Journal of Functional Morphology and Kinesiology ◽

10.3390/jfmk6020048 ◽

2021 ◽

Vol 6 (2) ◽

pp. 48

Author(s):

Elisa Innocenzi ◽

Ida Cariati ◽

Emanuela De Domenico ◽

Erika Tiberi ◽

Giovanna D’Arcangelo ◽

...

Keyword(s):

Alternative Splicing ◽

Aerobic Exercise ◽

Frontal Cortex ◽

Molecular Mechanisms ◽

Splice Variants ◽

Brain Regions ◽

Synaptic Function ◽

Molecular Changes ◽

Beneficial Effects ◽

The Impact

Aerobic exercise (AE) is known to produce beneficial effects on brain health by improving plasticity, connectivity, and cognitive functions, but the underlying molecular mechanisms are still limited. Neurexins (Nrxns) are a family of presynaptic cell adhesion molecules that are important in synapsis formation and maturation. In vertebrates, three-neurexin genes (NRXN1, NRXN2, and NRXN3) have been identified, each encoding for α and β neurexins, from two independent promoters. Moreover, each Nrxns gene (1–3) has several alternative exons and produces many splice variants that bind to a large variety of postsynaptic ligands, playing a role in trans-synaptic specification, strength, and plasticity. In this study, we investigated the impact of a continuous progressive (CP) AE program on alternative splicing (AS) of Nrxns on two brain regions: frontal cortex (FC) and hippocampus. We showed that exercise promoted Nrxns1–3 AS at splice site 4 (SS4) both in α and β isoforms, inducing a switch from exon-excluded isoforms (SS4−) to exon-included isoforms (SS4+) in FC but not in hippocampus. Additionally, we showed that the same AE program enhanced the expression level of other genes correlated with synaptic function and plasticity only in FC. Altogether, our findings demonstrated the positive effect of CP AE on FC in inducing molecular changes underlying synaptic plasticity and suggested that FC is possibly a more sensitive structure than hippocampus to show molecular changes.

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Targeting fibroblast growth factor receptors to combat aggressive ependymoma

Acta Neuropathologica ◽

10.1007/s00401-021-02327-x ◽

2021 ◽

Author(s):

Daniela Lötsch ◽

Dominik Kirchhofer ◽

Bernhard Englinger ◽

Li Jiang ◽

Konstantin Okonechnikov ◽

...

Keyword(s):

Growth Factor ◽

Fibroblast Growth Factor ◽

Molecular Mechanisms ◽

Radial Glia ◽

Growth Factor Receptors ◽

Dominant Negative ◽

Mrna Levels ◽

Fibroblast Growth ◽

Cell Models ◽

Fibroblast Growth Factor Receptors

AbstractEpendymomas (EPN) are central nervous system tumors comprising both aggressive and more benign molecular subtypes. However, therapy of the high-risk subtypes posterior fossa group A (PF-A) and supratentorial RELA-fusion positive (ST-RELA) is limited to gross total resection and radiotherapy, as effective systemic treatment concepts are still lacking. We have recently described fibroblast growth factor receptors 1 and 3 (FGFR1/FGFR3) as oncogenic drivers of EPN. However, the underlying molecular mechanisms and their potential as therapeutic targets have not yet been investigated in detail. Making use of transcriptomic data across 467 EPN tissues, we found that FGFR1 and FGFR3 were both widely expressed across all molecular groups. FGFR3 mRNA levels were enriched in ST-RELA showing the highest expression among EPN as well as other brain tumors. We further identified high expression levels of fibroblast growth factor 1 and 2 (FGF1, FGF2) across all EPN subtypes while FGF9 was elevated in ST-EPN. Interrogation of our EPN single-cell RNA-sequencing data revealed that FGFR3 was further enriched in cycling and progenitor-like cell populations. Corroboratively, we found FGFR3 to be predominantly expressed in radial glia cells in both mouse embryonal and human brain datasets. Moreover, we detected alternative splicing of the FGFR1/3-IIIc variant, which is known to enhance ligand affinity and FGFR signaling. Dominant-negative interruption of FGFR1/3 activation in PF-A and ST-RELA cell models demonstrated inhibition of key oncogenic pathways leading to reduced cell growth and stem cell characteristics. To explore the feasibility of therapeutically targeting FGFR, we tested a panel of FGFR inhibitors in 12 patient-derived EPN cell models revealing sensitivity in the low-micromolar to nano-molar range. Finally, we gain the first clinical evidence for the activity of the FGFR inhibitor nintedanib in the treatment of a patient with recurrent ST-RELA. Together, these preclinical and clinical data suggest FGFR inhibition as a novel and feasible approach to combat aggressive EPN.

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Role of glucocorticoid receptor and CCAAT/enhancer-binding protein α in the feed-forward induction of 11β-hydroxysteroid dehydrogenase type 1 expression by cortisol in human amnion fibroblasts

Journal of Endocrinology ◽

10.1677/joe-07-0303 ◽

2007 ◽

Vol 195 (2) ◽

pp. 241-253 ◽

Cited By ~ 54

Author(s):

Zhen Yang ◽

Chunming Guo ◽

Ping Zhu ◽

Wenjiao Li ◽

Leslie Myatt ◽

...

Keyword(s):

Glucocorticoid Receptor ◽

Mrna Expression ◽

Promoter Region ◽

Molecular Mechanisms ◽

Hydroxysteroid Dehydrogenase ◽

Dominant Negative ◽

Forward Induction ◽

Human Amnion ◽

Feed Forward

The amount of cortisol available to its receptors is increased by the pre-receptor enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which converts cortisone to cortisol. We examined the molecular mechanisms of the feedback effect of cortisol on 11β-HSD1 mRNA expression in human amnion fibroblasts. Our data showed that cortisol-induced 11β-HSD1 mRNA expression dose dependently in amnion fibroblasts, which could be completely blocked both by the mRNA transcription inhibitor 5,6-dichlorobenzimidazole riboside and by the glucocorticoid receptor (GR) antagonist RU486, and partially blocked by global inhibition of CCAAT/enhancer-binding proteins (C/EBPs) with transfection of C/EBP-specific dominant-negative expression CMV500 plasmid (AC/EBP) into the cells. Likewise, the induction of the promoter activity by cortisol could also be completely blocked by RU486 and partially by AC/EBP transfection. Progressive 5′ deletion of the 11β-HSD1promoter located the region responsible for cortisol’s induction within −204 bp upstream to the transcription start site. Specific nucleotide mutations of the putative glucocorticoid responsive element or CCAAT in this promoter region attenuated the induction by cortisol. Moreover, chromatin immunoprecipitation assay and electrophoretic mobility shift assay showed that GR and C/EBPα but not C/EBPβ could bind this promoter region upon cortisol stimulation of amnion fibroblasts. In conclusion, we demonstrated that GR and C/EBPα were involved in cortisol-induced 11β-HSD1 mRNA expression via binding to 11β-HSD1 promoter in amnion fibroblasts, which may cast a feed-forward production of cortisol in the fetal membranes at the end of gestation.

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Suppressor Mutations Bypass the Requirement of fluG for Asexual Sporulation and Sterigmatocystin Production in Aspergillus nidulans

Genetics ◽

10.1093/genetics/165.3.1083 ◽

2003 ◽

Vol 165 (3) ◽

pp. 1083-1093

Author(s):

Jeong-Ah Seo ◽

Yajun Guan ◽

Jae-Hyuk Yu

Keyword(s):

Aspergillus Nidulans ◽

Molecular Mechanisms ◽

Dominant Negative ◽

Wild Type ◽

Dominant Mutation ◽

Site Mutation ◽

Asexual Sporulation ◽

Dominant Negative Mutation ◽

Suppressor Mutations ◽

Early Developmental

Abstract Asexual sporulation (conidiation) in the filamentous fungus Aspergillus nidulans requires the early developmental activator fluG. Loss of fluG results in the blockage of both conidiation and production of the mycotoxin sterigmatocystin (ST). To investigate molecular mechanisms of fluG-dependent developmental activation, 40 suppressors of fluG (SFGs) that conidiate without fluG have been isolated and characterized. Genetic analyses showed that an individual suppression is caused by a single second-site mutation, and that all sfg mutations but one are recessive. Pairwise meiotic crosses grouped mutations to four loci, 31 of them to sfgA, 6 of them to sfgB, and 1 each to sfgC and sfgD, respectively. The only dominant mutation, sfgA38, also mapped to the sfgA locus, suggesting a dominant negative mutation. Thirteen sfgA and 1 sfgC mutants elaborated conidiophores in liquid submerged culture, indicating that loss of either of these gene functions not only bypasses fluG function but also results in hyperactive conidiation. While sfg mutants show varying levels of restored conidiation, all recovered the ability to produce ST at near wild-type levels. The fact that at least four loci are defined by recessive sfg mutations indicates that multiple genes negatively regulate conidiation downstream of fluG and that the activity of fluG is required to remove such repressive effects.

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Interaction of CREB and PGC-1α Induces Fibronectin Type III Domain-Containing Protein 5 Expression in C2C12 Myotubes

Cellular Physiology and Biochemistry ◽

10.1159/000494655 ◽

2018 ◽

Vol 50 (4) ◽

pp. 1574-1584 ◽

Cited By ~ 6

Author(s):

Xiu-ying Yang ◽

Margaret C.L. Tse ◽

Xiang Hu ◽

Wei-hua Jia ◽

Guan-hua Du ◽

...

Keyword(s):

Skeletal Muscle ◽

Molecular Mechanisms ◽

Metabolic Effects ◽

Hek293 Cells ◽

Metabolic Phenotype ◽

C2c12 Myotubes ◽

Type Iii ◽

Fibronectin Type Iii ◽

Fibronectin Type Iii Domain ◽

Fibronectin Type

Background/Aims: Fibronectin type III domain-containing protein 5 (FNDC5), also known as irisin, is a myokine secreted from muscle in response to exercise. However, the molecular mechanisms that regulate FNDC5 expression and the functional significance of irisn in skeletal muscle remain unknown. In this study, we explored the potential pathways that induce FNDC5 expression and delineated the metabolic effects of irisin on skeletal muscle. Methods: C2C12 myotubes were treated with drugs at various concentrations and durations. The expression and activation of genes were measured by real-time polymerase chain reaction (qRT-PCR) and Western blotting. Oxidative phosphorylation was quantified by measuring the oxygen consumption rate (OCR). Results: We found that the exercise-mimicking treatment (cAMP, forskolin and isoproterenol) increased Fndc5 expression in C2C12 myotubes. CREB over-expressed C2C12 myotubes displayed higher Fndc5 expression. CREB over-expression also promoted peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) expression. PGC-1α-induced Fndc5 expression was blocked when the dominant negative form of CREB (S133A) was present. PGC-1α mutation (S570A) also decreased Fndc5 expression. Immunoprecipitation showed that overexpressed PGC-1α complexed with CREB in HEK293 cells. C2C12 myotubes treated with forskolin also increased endogenous CREB and PGC-1α binding. Functionally, irisin treatment increased mitochondrial respiration, enhanced ATP production, promoted fatty acid oxidation but decreased glycolysis in myotubes. Conclusion: Our observation indicates that cAMP-mediated PGC-1α/CREB interaction triggers Fndc5 expression, which acts as an autocrine/paracrine to shape the metabolic phenotype of myotubes.

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