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Guy Smagghe - One of the best experts on this subject based on the ideXlab platform.
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Cloning and functional analysis of the Ecdysteroid Receptor complex in the opossum shrimp Neomysis integer (Leach, 1814).
Aquatic Toxicology, 2013Co-Authors: R. De Wilde, Thomas Soin, Luc Swevers, Pierre Rouge, K Cooreman, Olivier Christiaens, Colin R. Janssen, Guy SmaggheAbstract:a b s t r a c t In this paper, the non-target effects of tebufenozide were evaluated on the estuarine crustacean, the opposum shrimp Neomysis integer (Leach, 1814). Tebufenozide is a synthetic non-steroidal ecdysone agonist insecticide and regarded as potential endocrine-disrupting chemical (EDC). N. integer is the most used crustacean in ecotoxicological research in parallel to Daphnia sp. and has been proposed for the regulatory testing of potential EDCs in the US, Europe and Japan. Major results were: (i) cDNAs encoding the Ecdysteroid Receptor (EcR) and the retinoid-X-Receptor (RXR), were cloned and sequenced, and subsequent molecular phylogenetic analysis (maximum likeli- hood and neighbor-joining) revealed that the amino acid sequence of the ligand binding domain (LBD) of N. integer EcR (NiEcR) clusters as an outgroup of the Crustacea, while NiRXR-LBD clusters in the Malacos- tracan clade (bootstrap percentage = 75%). (ii) 3D-modeling of ligand binding to NiEcR-LBD demonstrated an incompatibility of the insecticide tebufenozide to fit into the NiEcR-ligand binding pocket. This was in great contrast to ponasterone A (PonA) that is the natural molting hormone in Crustacea and for which efficient docking was demonstrated. In addition, the heterodimerization of NiEcR-LBD with the com- mon shrimp Crangon crangon (Linnaeus, 1758) RXR-LBD (CrcRXR-LBD) was also modeled in silico. (iii) With use of insect Hi5 cells, chimeric constructs of NiEcR-LBD and CrcRXR-LBD fused to either the yeast Gal4-DNA binding domain (DBD) or Gal4-activation domain (AD) were cloned into expression plasmids and co-transfected with a Gal4 reporter to quantify the protein-protein interactions of NiEcR-LBD with CrcRXR-LBD. Investigation of the ligand effect of PonA and tebufenozide revealed that only the presence of PonA could induce dimerization of this heterologous Receptor complex. (iv) Finally, in an in vivo toxicity assay, N. integer juveniles were exposed to tebufenozide at a concentration of 100 g/L, and no effects against the molting process and nymphal development were scored.
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Ecdysteroid Receptor docking suggests that dibenzoylhydrazine-based insecticides are devoid of any deleterious effect on the parasitic wasp Psyttalia concolor (Hym. Braconidae)
Pest management science, 2012Co-Authors: Paloma Bengochea, Pierre Rouge, Olivier Christiaens, Fermín Amor, Elisa Viñuela, Pilar Medina, Guy SmaggheAbstract:BACKGROUND: The moulting accelerating compounds (MACs) or Ecdysteroid agonists represent a selective group of insecticides acting upon binding to the Ecdysteroid Receptor (EcR) and leading to lethal premature moulting in larval stages and aborted reproduction in adults. Psyttalia concolor Szepl. is a useful parasitic wasp attacking important tephritid pests such as the medfly and olive fruit fly. RESULTS: Contact and oral exposure in the laboratory of female parasitic wasps to the dibenzoylhydrazine-based methoxyfenozide, tebufenozide and RH-5849 did not provoke negative effects. No mortality and no reduction in beneficial capacity were observed. The ligand-binding domain (LBD) of the EcR of P. concolor was sequenced, and a homology protein model was constructed which confirmed a cavity structure with 12 α-helices, harbouring the natural insect moulting hormone 20-hydroxyecdysone. However, a steric clash occurred for the MAC insecticides owing to a restricted extent of the ligand-binding cavity of the PcLBD-EcR, while they did dock well in that of susceptible insects. CONCLUSIONS: The insect toxicity assays demonstrated that MACs are selective for P. concolor. The modelling/docking experiments are indications that these insecticides do not bind with the LBD-EcR of P. concolor and support the theory that they show no biological effects in the parasitic wasp. These data may help in explaining the compatible use of MACs together with parasitic wasps in IPM programmes. Copyright © 2012 Society of Chemical Industry
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Saponins do not affect the Ecdysteroid Receptor complex but cause membrane permeation in insect culture cell lines.
Journal of insect physiology, 2011Co-Authors: Ellen De Geyter, Thomas Soin, Luc Swevers, Danny Geelen, Guy SmaggheAbstract:Abstract This project studied the effects of four saponins with a triterpenoid ( Quillaja saponaria saponin and aescin) or steroid structure (digitonin and diosgenin which is the deglycosylated form of dioscin) on insect cells, namely Schneider S2 cells of Drosophila melanogaster (Diptera). A series of different experiments were performed to investigate potential mechanisms of action by saponins with regard to Ecdysteroid Receptor (EcR) responsiveness, cell viability, cell membrane permeation, and induction of apoptosis with DNA fragmentation and caspase-3 like activity. Major results were that (1) exposure of S2 cells containing an EcR-based reporter construct to a concentration series of each saponin scored no EcR activation, while (2) a loss of Ecdysteroid signaling was observed with median inhibitory concentrations (IC 50 ’s) of 3–50 μM, and in parallel (3) a concentration-dependent change in loss of cell numbers in an cell viability assay with median effective concentrations (EC 50 ’s) of 8–699 μM. In continuation, it was of interest that (4) a trypan blue assay with Q. saponaria saponin confirmed the cell membrane permeation effect leading to cell toxicity with a median lethal concentration (LC 50 ) value of 44 μM, and interestingly this effect was very rapid. Another three interesting observations were that (5) exposure to 20E at 500 nM as used in the EcR-based report assay induced caspase-3 like activities which may help to explain the discrepancies between loss of EcR-responsiveness and cell viability, (6) low concentrations of saponins induced DNA fragmentation and caspase-3 like activities, confirming their potential to induce apoptosis, and (7) the saponin effects were counteracted with addition of cholesterol to the culture medium. In general the data obtained provide evidence that the anti-Ecdysteroid action by saponins is not based on a true antagonistic interaction with EcR signaling, but can be explained by a cytotoxic action due to permeation of the insect cell membrane.
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the heterodimeric Ecdysteroid Receptor complex in the brown shrimp crangon crangon ecr and rxr isoform characteristics and sensitivity towards the marine pollutant tributyltin
General and Comparative Endocrinology, 2011Co-Authors: Yves Verhaegen, Luc Swevers, K Parmentier, Pierre Rouge, Wim De Coen, K Cooreman, Ellen Renders, Guy SmaggheAbstract:Decapod crustaceans are characterized by multiple Ecdysteroid Receptor (EcR) and retinoid-X-Receptor (RXR) isoforms, which likely exhibit variant dimerization and transactivation interactions. In the brown shrimp C. crangon we cloned C-terminally truncated CrcEcR and CrcRXR isoforms and isoforms exhibiting deletions within the hinge region. For the former, in silico modeling of the CrcEcR indicated that, where the conserved helices H10 and H11 of the ligand-binding domain (LBD) are missing, an alternative C-terminal a-helix repairs the ligand-binding pocket (LBP). The truncated CrcRXR isoforms lack a major part of the LBD (H4–H12), thereby compromising ligand binding and dimerization. Through an in vitro Ecdysteroid responsive reporter assay, we showed that these natural Receptor variations do not impair Receptor functioning but probably alter the Receptor dimerization preferences. By the same in vitro assay, using full-length CrcEcR and CrcRXR, the effect of tributyltin (TBT) on Ecdysteroid-induced transactivation was evaluated. The transactivation by 10 nM PonA was reduced with 64% by 20 nM TBT. In silico modeling confirmed that TBT fits in the full-length CrcRXR–LBD. Furthermore, semi-quantitative PCR indicated altered expression of CrcEcR and CrcRXR isoforms after in vivo acute exposure to TBT, especially in the ovaries.
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the brown shrimp crangon crangon l Ecdysteroid Receptor complex cloning structural modeling of the ligand binding domain and functional expression in an ecr deficient drosophila cell line
General and Comparative Endocrinology, 2010Co-Authors: Yves Verhaegen, Thomas Soin, Luc Swevers, K Parmentier, Pierre Rouge, Wim De Coen, K Cooreman, Guy SmaggheAbstract:cDNAs encoding Ecdysteroid Receptor (EcR) and retinoid X Receptor (RXR) were cloned and sequenced from brown shrimp Crangon crangon (Crustacea: Decapoda), a common faunal species and commercially important in the North-West European coastal waters. A 3D model of the ligand-binding domain (LBD) of EcR was created and docking of ponasterone A (PonA) was simulated in silico. Finally, we report the transfection of expression plasmids for these Receptors in the mutant Drosophila L57-3-11 cell line. Through an Ecdysteroid responsive reporter assay we clearly prove the functionality of shrimp Ecdysteroid Receptor in the transfected L57-3-11 cell line. Our results indicate that the Drosophila L57-3-11 cell line and in silico LBD modeling can be used to study the function of crustacean Ecdysteroid Receptors and be applied to assess endocrine disrupting effects on non-target crustacean species.
Margarethe Spindler-barth - One of the best experts on this subject based on the ideXlab platform.
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Biological effects of muristerone A and turkesterone on the epithelial cell line from Chironomus tentans (Diptera: Chironomidae) and correlation with binding affinity to the Ecdysteroid Receptor
European Journal of Entomology, 2013Co-Authors: Margarethe Spindler-barth, S Quack, P Rauch, Spindler K.-d.Abstract:Ecdysteroids, which possess an alpha-hydroxyl group at C11, bind with about the same affinity to the Ecdysteroid Receptor as compared to the corresponding compounds lacking the hydroxyl group. This is shown for turkesterone/20-hydroxyecdysone and muristerone A using Receptor preparations from the epithelial cell line from Chironomus tentans. For all compounds tested, the potency to elicit a hormonal response corresponds with the affinity to the Ecdysteroid Receptor as determined by induction of acetylcholinesterase, a marker enzyme for cell differentiation.
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N- and C-terminal degradation of Ecdysteroid Receptor isoforms, when transiently expressed in mammalian CHO cells, is regulated by the proteasome and cysteine and threonine proteases
Insect molecular biology, 2012Co-Authors: S. Schauer, Timo Burster, Margarethe Spindler-barthAbstract:Transcriptional activity of nuclear Receptors is the result of transactivation capability and the concentration of the Receptor protein. The concentration of Ecdysteroid Receptor (EcR) isoforms, constitutively expressed in mammalian CHO cells, is dependent on a number of factors. As shown previously, ligand binding stabilizes Receptor protein concentration. In this paper, we investigate the degradation of EcR isoforms and provide evidence that N-terminal degradation is modulated by isoform-specific ubiquitination sites present in the A/B domains of EcR-A and -B1. This was demonstrated by the increase in EcR concentration by treatment with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), an inhibitor of ubiquitin-mediated proteasomal degradation and by deletion of ubiquitination sites. In addition, EcR is degraded by the peptidyl-dipeptidase cathepsin B (CatB) and the endopeptidase cathepsin S (CatS) at the C-terminus in an isoform-specific manner, despite identical C-termini. Ubiquitin-proteasome-mediated degradation and the proteolytic action are modulated by heterodimerization with Ultraspiracle (USP). The complex regulation of Receptor protein concentration offers an additional opportunity to regulate transcriptional activity in an isoform- and target cell-specific way and allows the temporal limitation of hormone action.
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Influence of hormone response elements (HREs) on Ecdysteroid Receptor concentration
Insect molecular biology, 2011Co-Authors: S. Schauer, Anca Azoitei, S. Braun, Margarethe Spindler-barthAbstract:Transcriptional activity of nuclear Receptors is the result of transactivation capability and Receptor protein concentration. The concentration of Ecdysteroid Receptor (EcR) constitutively expressed in vertebrate cells varies depending on the isoforms. Besides ligand binding and heterodimerization with ultraspiracle (USP), which stabilizes Receptor protein concentration, degradation is regulated by interaction of the Receptor complex with different Ecdysteroid response elements (EcREs). Coexpression of EcREs significantly reduces Ecdysteroid Receptor concentration depending on the type of EcRE. Transcriptional activity and interaction with hormone response elements (HREs) as determined by Electrophoretic Mobility Shift Assay (EMSA) are often inversely related to Receptor protein concentration. The complex regulation of Receptor protein concentration offers an additional opportunity to regulate transcriptional activity in an isoform- and target cell-specific manner and allows the temporal limitation of hormone action.
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The N-terminus of Ecdysteroid Receptor isoforms and ultraspiracle interacts with different Ecdysteroid response elements in a sequence specific manner to modulate transcriptional activity.
The Journal of steroid biochemistry and molecular biology, 2011Co-Authors: S. Schauer, Vincent C. Henrich, Jenna Callender, Margarethe Spindler-barthAbstract:The functional insect Ecdysteroid Receptor is comprised of two nuclear Receptors, the Ecdysteroid Receptor (EcR) and the RXR homologue, ultraspiracle (USP), which form a heterodimer. The dimer recognizes various hormone response elements and the effect of these elements on transcriptional activity of EcR isoforms was determined in vertebrate cells transfected with EcR and USP. Only constitutive activity mediated by the core response elements was preserved after elimination of nonspecific binding sites on the DNA of the vector. The constitutive transcriptional activity was regulated in a complex manner by the N-termini of both EcR and USP, the DBD of USP and the type and number of hormone response elements (HRE). Cooperative effects at oligomeric response elements particularly DR1 depended on the type of Ecdysteroid response element and the N-termini of EcR and USP. The DBD of USP abolishes or attenuates synergistic effects. The data show that in the absence of hormone, transcriptional activity is regulated in a complex manner that offers additional possibilities for Ecdysteroid Receptor mediated gene regulation during development.
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The importance of exportin and Ran for nucleocytoplasmic shuttling of the Ecdysteroid Receptor.
Archives of insect biochemistry and physiology, 2010Co-Authors: Katarzyna Betanska, Margarethe Spindler-barth, Celine Hönl, Klaus-dieter SpindlerAbstract:Nuclear localization of the Ecdysteroid Receptor (EcR) is increased in HeLa cells if exportin-1 (CRM1), a predominant carrier for export of proteins and RNA from the nucleus into the cytoplasm, is knocked down by siRNA against exportin. However, knockdown of the small G protein Ran, which is essential for nuclear transport, leads to an arrest of EcR in the cytoplasm, but does not prevent efficient nuclear import of the most important heterodimerization partner of EcR, ultraspiracle (Usp). Like in vertebrate cells, EcR is also distributed heterogeneously in Drosophila melanogaster S2 cells but shifted exclusively to the nucleus, if Usp is present. © 2010; Wiley Periodicals, Inc.
Margarethe Spindlerbarth - One of the best experts on this subject based on the ideXlab platform.
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influence of cell cycle on Ecdysteroid Receptor in cho k1 cells
Archives of Insect Biochemistry and Physiology, 2009Co-Authors: Katarzyna Betanska, Margarethe Spindlerbarth, Susan Czogalla, Klaus-dieter SpindlerAbstract:CHO-K1 cells are routinely used for characterization of ecdysone Receptor (EcR) function, because these vertebrate cells are devoid of endogenous ecdysone Receptor protein. Moreover, the endogenous expression of RXR, the vertebrate orthologue of Ultraspiracle (Usp), the most important heterodimerization partner, is neglectable. In contrast to insect cells, there is also no influence of moulting hormone on CHO-K1 cells on cell proliferation either in the absence or presence of transiently expressed EcR. In contrast to Usp, which is exclusively found in nuclei, EcR is heterogeneously distributed between cytoplasm and nuclei in non-synchronized cells. Synchronization of CHO-K1 cells by nocodazole revealed that the cell cycle influences Receptor concentration with lowest amounts in late S-phase and G2/M phase and intracellular distribution of the Receptor protein showing a minimum of Receptors present in nuclei during S-phase. EcR, but not Usp reduces cyclin D1 expression and cyclin D1 concentration is impaired by cyclin D1. Coimmunoprecipitation studies reveal physical interaction of EcR and cyclin D1.
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properties of Ecdysteroid Receptors from diverse insect species in a heterologous cell culture system a basis for screening novel insecticidal candidates
FEBS Journal, 2009Co-Authors: Joshua M. Beatty, Guy Smagghe, Yoshiaki Nakagawa, Margarethe Spindlerbarth, Takehiko Ogura, Vincent C. HenrichAbstract:Insect development is driven by the action of Ecdysteroids on morphogenetic processes. The classic Ecdysteroid Receptor is a protein heterodimer composed of two nuclear Receptors, the ecdysone Receptor (EcR) and Ultraspiracle (USP), the insect ortholog of retinoid X Receptor. The functional properties of EcR and USP vary among insect species, and provide a basis for identifying novel and species-specific insecticidal candidates that disrupt this Receptor’s normal activity. A heterologous mammalian cell culture assay was used to assess the transcriptional activity of the heterodimeric Ecdysteroid Receptor from species representing two major insect orders: the fruit fly, Drosophila melanogaster (Diptera), and the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera). Several nonsteroidal agonists evoked a strong response with the L. decemlineata heterodimer that was consistent with biochemical and in vivo evidence, whereas the D. melanogaster Receptor’s response was comparatively modest. Conversely, the phytoEcdysteroid muristerone A was more potent with the D. melanogaster heterodimer. The additional presence of juvenile hormone III potentiated the inductive activity of muristerone A in the Receptors from both species, but juvenile hormone III was unable to potentiate the inductive activity of the diacylhydrazine methoxyfenozide (RH2485) in the Receptor of either species. The effects of USP on Ecdysteroid-regulated transcriptional activity also varied between the two species. When it was tested with D. melanogaster EcR isoforms, basal activity was lower and ligand-dependent activity was higher with L. decemlineata USP than with D. melanogaster USP. Generally, the species-based differences validate the use of the cell culture assay screen for novel agonists and potentiators as species-targeted insecticidal candidates.
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functional analysis of Ecdysteroid Receptor from drosophila melanogaster in vitro
2009Co-Authors: Anca Azoitei, S. Braun, Heike Ruff, Christian Tremmel, Margarethe SpindlerbarthAbstract:Ecdysone Receptor (EcR) was expressed in vertebrate cells to study its functional properties in the absence of the heterodimerization partner Ultraspiracle (Usp) and to avoid interference with endogenous Receptor isoforms. Comparison of different isoforms affords determination of Receptor concentration, which was achieved either by determination of ligand binding sites by Scatchard analysis or by quantitative evaluation of specific Western blot signals, but not by normalization on transfection efficiency as determined by cotransfection with a constitutive reporter plasmid. Ligand- and DNA- binding, and transcriptional activity of EcR isoforms and the influence of Ultraspiracle (Usp) were described.
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expression of Ecdysteroid Receptor and ultraspiracle from chironomus tentans insecta diptera in e coli and purification in a functional state
Insect Biochemistry and Molecular Biology, 2002Co-Authors: Marco Grebe, Margarethe SpindlerbarthAbstract:Full length clones of Ecdysteroid Receptor (EcR) and Ultraspiracle (USP) from Chironomus tentans were expressed as GST fusion proteins in E. coli and purified by affinity chromatography. The absence of detergents during the purification procedure is essential for retaining Receptor function, especially ligand binding. Presence of USP is mandatory for ligand binding to EcR, but no other cofactors or posttranslational modifications seem to be important, since Scatchard plots revealed the same characteristics (two high affinity binding sites for Ponasterone A with K(D1)=0.24+/-0.1nM and K(D2)=3.9+/-1.3.nM) as found in 0.4 M NaCl extracts of Chironomus cells. Gel mobility shift assays showed binding of the heterodimer to PAL and DR5 even after removal of the GST-tag, whereas EcR binding to PAL1 is GST-dependent. USP binds preferentially to DR5. Addition of unprogrammed reticulocyte lysate improves ligand binding only slightly. Removal of GST has no effect on (3)H-ponasterone A binding, but alters DNA binding characteristics. Calculation of specific binding (5.3+3.0 nmol/mg GST EcR) revealed that 47+/-26% of purified Receptor protein was able to bind ligand. The addition of purified EcR to cell extracts of hormone resistant subclones of the epithelial cell line from C. tentans, which have lost their ability to bind ligand, restores specific binding of (3)H-ponasterone A.
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characterization of subclones of the epithelial cell line from chironomus tentans resistant to the insecticide rh 5992 a non steroidal moulting hormone agonist
Insect Biochemistry and Molecular Biology, 2000Co-Authors: Marco Grebe, Peter Rauch, Margarethe SpindlerbarthAbstract:Selection of hormone resistant subclones in the continuous presence of the insecticide and Ecdysteroid mimick RH 5992 (tefubenozide) resulted preferentially in clones with defects in Ecdysteroid Receptor function. RH 5992 is already degraded to polar products in wild-type cells; no increase in metabolism of tefubenozide is observed in resistant clones. According to Western blots, Ecdysteroid Receptor (EcR) and its heterodimerization partner ultraspiracle (USP) are present in all resistant clones. The concentrations are comparable to wild-type cells, but in three clones the extent of phosphorylation of USP is diminished. With regard to hormone binding several types of hormone resistance are distinguished: (1) The same two high-affinity hormone recognition sites are present as in wild-type cells (KD1=0.31∠0.28 nM, KD2=6.5∠2.4 nM) but the number of binding sites is reduced. (2) The binding site with the lower affinity (KD2) is missing. (3) The binding site with the higher affinity (KD1) is missing. (4) No specific binding is observed. Ponasterone A binding can be rescued by addition of EcR but not by USP. (5) Ligand specificity is altered. RH 5992 can not compete [ 3 H]-ponasterone A as efficient as in wild-type cells. © 2000 Elsevier Science Ltd. All rights reserved.
Luc Swevers - One of the best experts on this subject based on the ideXlab platform.
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Cloning and functional analysis of the Ecdysteroid Receptor complex in the opossum shrimp Neomysis integer (Leach, 1814).
Aquatic Toxicology, 2013Co-Authors: R. De Wilde, Thomas Soin, Luc Swevers, Pierre Rouge, K Cooreman, Olivier Christiaens, Colin R. Janssen, Guy SmaggheAbstract:a b s t r a c t In this paper, the non-target effects of tebufenozide were evaluated on the estuarine crustacean, the opposum shrimp Neomysis integer (Leach, 1814). Tebufenozide is a synthetic non-steroidal ecdysone agonist insecticide and regarded as potential endocrine-disrupting chemical (EDC). N. integer is the most used crustacean in ecotoxicological research in parallel to Daphnia sp. and has been proposed for the regulatory testing of potential EDCs in the US, Europe and Japan. Major results were: (i) cDNAs encoding the Ecdysteroid Receptor (EcR) and the retinoid-X-Receptor (RXR), were cloned and sequenced, and subsequent molecular phylogenetic analysis (maximum likeli- hood and neighbor-joining) revealed that the amino acid sequence of the ligand binding domain (LBD) of N. integer EcR (NiEcR) clusters as an outgroup of the Crustacea, while NiRXR-LBD clusters in the Malacos- tracan clade (bootstrap percentage = 75%). (ii) 3D-modeling of ligand binding to NiEcR-LBD demonstrated an incompatibility of the insecticide tebufenozide to fit into the NiEcR-ligand binding pocket. This was in great contrast to ponasterone A (PonA) that is the natural molting hormone in Crustacea and for which efficient docking was demonstrated. In addition, the heterodimerization of NiEcR-LBD with the com- mon shrimp Crangon crangon (Linnaeus, 1758) RXR-LBD (CrcRXR-LBD) was also modeled in silico. (iii) With use of insect Hi5 cells, chimeric constructs of NiEcR-LBD and CrcRXR-LBD fused to either the yeast Gal4-DNA binding domain (DBD) or Gal4-activation domain (AD) were cloned into expression plasmids and co-transfected with a Gal4 reporter to quantify the protein-protein interactions of NiEcR-LBD with CrcRXR-LBD. Investigation of the ligand effect of PonA and tebufenozide revealed that only the presence of PonA could induce dimerization of this heterologous Receptor complex. (iv) Finally, in an in vivo toxicity assay, N. integer juveniles were exposed to tebufenozide at a concentration of 100 g/L, and no effects against the molting process and nymphal development were scored.
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Saponins do not affect the Ecdysteroid Receptor complex but cause membrane permeation in insect culture cell lines.
Journal of insect physiology, 2011Co-Authors: Ellen De Geyter, Thomas Soin, Luc Swevers, Danny Geelen, Guy SmaggheAbstract:Abstract This project studied the effects of four saponins with a triterpenoid ( Quillaja saponaria saponin and aescin) or steroid structure (digitonin and diosgenin which is the deglycosylated form of dioscin) on insect cells, namely Schneider S2 cells of Drosophila melanogaster (Diptera). A series of different experiments were performed to investigate potential mechanisms of action by saponins with regard to Ecdysteroid Receptor (EcR) responsiveness, cell viability, cell membrane permeation, and induction of apoptosis with DNA fragmentation and caspase-3 like activity. Major results were that (1) exposure of S2 cells containing an EcR-based reporter construct to a concentration series of each saponin scored no EcR activation, while (2) a loss of Ecdysteroid signaling was observed with median inhibitory concentrations (IC 50 ’s) of 3–50 μM, and in parallel (3) a concentration-dependent change in loss of cell numbers in an cell viability assay with median effective concentrations (EC 50 ’s) of 8–699 μM. In continuation, it was of interest that (4) a trypan blue assay with Q. saponaria saponin confirmed the cell membrane permeation effect leading to cell toxicity with a median lethal concentration (LC 50 ) value of 44 μM, and interestingly this effect was very rapid. Another three interesting observations were that (5) exposure to 20E at 500 nM as used in the EcR-based report assay induced caspase-3 like activities which may help to explain the discrepancies between loss of EcR-responsiveness and cell viability, (6) low concentrations of saponins induced DNA fragmentation and caspase-3 like activities, confirming their potential to induce apoptosis, and (7) the saponin effects were counteracted with addition of cholesterol to the culture medium. In general the data obtained provide evidence that the anti-Ecdysteroid action by saponins is not based on a true antagonistic interaction with EcR signaling, but can be explained by a cytotoxic action due to permeation of the insect cell membrane.
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the heterodimeric Ecdysteroid Receptor complex in the brown shrimp crangon crangon ecr and rxr isoform characteristics and sensitivity towards the marine pollutant tributyltin
General and Comparative Endocrinology, 2011Co-Authors: Yves Verhaegen, Luc Swevers, K Parmentier, Pierre Rouge, Wim De Coen, K Cooreman, Ellen Renders, Guy SmaggheAbstract:Decapod crustaceans are characterized by multiple Ecdysteroid Receptor (EcR) and retinoid-X-Receptor (RXR) isoforms, which likely exhibit variant dimerization and transactivation interactions. In the brown shrimp C. crangon we cloned C-terminally truncated CrcEcR and CrcRXR isoforms and isoforms exhibiting deletions within the hinge region. For the former, in silico modeling of the CrcEcR indicated that, where the conserved helices H10 and H11 of the ligand-binding domain (LBD) are missing, an alternative C-terminal a-helix repairs the ligand-binding pocket (LBP). The truncated CrcRXR isoforms lack a major part of the LBD (H4–H12), thereby compromising ligand binding and dimerization. Through an in vitro Ecdysteroid responsive reporter assay, we showed that these natural Receptor variations do not impair Receptor functioning but probably alter the Receptor dimerization preferences. By the same in vitro assay, using full-length CrcEcR and CrcRXR, the effect of tributyltin (TBT) on Ecdysteroid-induced transactivation was evaluated. The transactivation by 10 nM PonA was reduced with 64% by 20 nM TBT. In silico modeling confirmed that TBT fits in the full-length CrcRXR–LBD. Furthermore, semi-quantitative PCR indicated altered expression of CrcEcR and CrcRXR isoforms after in vivo acute exposure to TBT, especially in the ovaries.
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the brown shrimp crangon crangon l Ecdysteroid Receptor complex cloning structural modeling of the ligand binding domain and functional expression in an ecr deficient drosophila cell line
General and Comparative Endocrinology, 2010Co-Authors: Yves Verhaegen, Thomas Soin, Luc Swevers, K Parmentier, Pierre Rouge, Wim De Coen, K Cooreman, Guy SmaggheAbstract:cDNAs encoding Ecdysteroid Receptor (EcR) and retinoid X Receptor (RXR) were cloned and sequenced from brown shrimp Crangon crangon (Crustacea: Decapoda), a common faunal species and commercially important in the North-West European coastal waters. A 3D model of the ligand-binding domain (LBD) of EcR was created and docking of ponasterone A (PonA) was simulated in silico. Finally, we report the transfection of expression plasmids for these Receptors in the mutant Drosophila L57-3-11 cell line. Through an Ecdysteroid responsive reporter assay we clearly prove the functionality of shrimp Ecdysteroid Receptor in the transfected L57-3-11 cell line. Our results indicate that the Drosophila L57-3-11 cell line and in silico LBD modeling can be used to study the function of crustacean Ecdysteroid Receptors and be applied to assess endocrine disrupting effects on non-target crustacean species.
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Juvenile hormone analogs do not affect directly the activity of the Ecdysteroid Receptor complex in insect culture cell lines.
Journal of insect physiology, 2007Co-Authors: Thomas Soin, Luc Swevers, Hadi Mosallanejad, Rodica Efrose, Vassiliki Labropoulou, Kostas Iatrou, Guy SmaggheAbstract:During insect development, Ecdysteroids and juvenile hormones (JHs) interact to regulate larval growth, metamorphosis and reproduction but the molecular mechanisms by which both hormones influence each other's activity remain unknown. Because of their ease of use and straightforward genetic manipulation, insect cell lines often have been used to clarify the actions and interactions of hormones at the molecular level. Here we report on the use of two insect culture cell lines, Drosophila melanogaster S2 and Bombyx mori Bm5 cells, to investigate two molecular processes in which Ecdysteroids and JH have been shown to interact: (1) direct modulation of the activity of the Ecdysteroid Receptor transcription complex and (2) interference at the level of induction of the primary gene E75. Our data do not support JH analogs (JHAs) acting through the above processes: 'antagonism' of Ecdysteroid Receptor activity by JHAs correlated with cytotoxicity and induction of E75 expression by JHAs was not demonstrated. However, we confirm previous studies in which it was observed that methoprene can partially reverse the growth inhibition by 20E in S2 cells (but not Bm5 cells). Therefore, the molecular mechanism by which both hormones influence each other's activity to regulate cell growth in S2 cells remains unknown.
Klaus-dieter Spindler - One of the best experts on this subject based on the ideXlab platform.
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The importance of exportin and Ran for nucleocytoplasmic shuttling of the Ecdysteroid Receptor.
Archives of insect biochemistry and physiology, 2010Co-Authors: Katarzyna Betanska, Margarethe Spindler-barth, Celine Hönl, Klaus-dieter SpindlerAbstract:Nuclear localization of the Ecdysteroid Receptor (EcR) is increased in HeLa cells if exportin-1 (CRM1), a predominant carrier for export of proteins and RNA from the nucleus into the cytoplasm, is knocked down by siRNA against exportin. However, knockdown of the small G protein Ran, which is essential for nuclear transport, leads to an arrest of EcR in the cytoplasm, but does not prevent efficient nuclear import of the most important heterodimerization partner of EcR, ultraspiracle (Usp). Like in vertebrate cells, EcR is also distributed heterogeneously in Drosophila melanogaster S2 cells but shifted exclusively to the nucleus, if Usp is present. © 2010; Wiley Periodicals, Inc.
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Ecdysteroid hormone action
Cellular and Molecular Life Sciences, 2009Co-Authors: Klaus-dieter Spindler, S. Braun, C. Hönl, Ch. Tremmel, H. Ruff, M. Spindler-barthAbstract:Several reviews devoted to various aspects of ecdysone research have been published during the last few years. Therefore, this article concentrates mainly on the considerable progress in ecdysone research observed recently, and will cover the results obtained during the last 2 years. The main emphasis is put on the molecular mode of Ecdysteroid Receptor-mediated hormone action. Two examples of interaction with other hormonal signalling pathways are described, namely crosstalk with juvenile hormone and insulin. Some selected, recently investigated examples of the multitude of hormonal responses are described. Finally, ecological aspects and some practical applications are discussed.
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influence of cell cycle on Ecdysteroid Receptor in cho k1 cells
Archives of Insect Biochemistry and Physiology, 2009Co-Authors: Katarzyna Betanska, Margarethe Spindlerbarth, Susan Czogalla, Klaus-dieter SpindlerAbstract:CHO-K1 cells are routinely used for characterization of ecdysone Receptor (EcR) function, because these vertebrate cells are devoid of endogenous ecdysone Receptor protein. Moreover, the endogenous expression of RXR, the vertebrate orthologue of Ultraspiracle (Usp), the most important heterodimerization partner, is neglectable. In contrast to insect cells, there is also no influence of moulting hormone on CHO-K1 cells on cell proliferation either in the absence or presence of transiently expressed EcR. In contrast to Usp, which is exclusively found in nuclei, EcR is heterogeneously distributed between cytoplasm and nuclei in non-synchronized cells. Synchronization of CHO-K1 cells by nocodazole revealed that the cell cycle influences Receptor concentration with lowest amounts in late S-phase and G2/M phase and intracellular distribution of the Receptor protein showing a minimum of Receptors present in nuclei during S-phase. EcR, but not Usp reduces cyclin D1 expression and cyclin D1 concentration is impaired by cyclin D1. Coimmunoprecipitation studies reveal physical interaction of EcR and cyclin D1.
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Impact of heterodimerization on intracellular localization of the Ecdysteroid Receptor (EcR).
Archives of insect biochemistry and physiology, 2008Co-Authors: Claudia Nieva, Margarethe Spindler-barth, Klaus-dieter SpindlerAbstract:Initially, nuclear import of the Ecdysteroid Receptor (EcR) in vertebrate cells (CHO-K1 and COS-7) does not afford a heterodimerization partner. Later on, EcR is retained in the nucleus only in the presence of a heterodimerization partner. Ultraspiracle (Usp) is more efficient compared to its vertebrate orthologue RXR and leads to an exclusively nuclear localization of EcR even in the absence of ligand. The DNA binding domain of the heterodimerization partner is important for retainment of EcR in the nucleus as shown by Usp4 (Usp(R130C)), which has lost its DNA binding capability. The C-terminal end of Usp (Usp(Delta205-508)) encompassing the C-terminal part of the D-domain and the E- and F-domains are essential for retainment of EcR in the nucleus. Nuclear localization is further influenced by cell-specific factors, since hormone and heterodimerization stabilizes the EcR protein in a cell-specific way.
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Influence of hormone on intracellular localization of the Drosophila melanogaster Ecdysteroid Receptor (EcR).
Cellular signalling, 2007Co-Authors: Claudia Nieva, Anca Azoitei, Margarethe Spindler-barth, Klaus-dieter SpindlerAbstract:Abstract In the absence of hormone the Ecdysteroid Receptor (EcR) is distributed between the cytoplasm and the nucleus. Addition of the hormone muristerone A increases nuclear localization of wild type EcR within 5–10 min. Mutation of M504 to alanine, an amino acid, which is essential for ligand binding and which is situated in helix 5 of the ligand binding domain, abolishes hormone binding but still allows nuclear localization at only slightly reduced levels in the absence of hormone, whereas nuclear localization of EcRM504R is nearly abolished. Cotransfection with ultraspiracle (USP), the invertebrate ortholog of RXR, leads to exclusively nuclear localization of wild type EcR and EcRM504A indicating that basal heterodimerization in the absence of hormone is still possible. In the presence of Usp, EcRM504R is only partially localized in the nucleus. EMSA experiments show that the ligand muristerone A enhances binding of wild type EcR, but only slighthly of mutated EcRs, to the canonical hsp 27 ecdysone response element. This is confirmed by transactivation studies. The results indicate that the architecture of the E-domain of EcR is important for nuclear localization even in the absence of a ligand.