The morphological response of Kc-H cells to ecdysteroids: Hormonal specificity

Summary Cells of the line Kc, derived fromDrosophila melanogaster embryos, extend long processes when exposed to ecdysteroid hormones. We have devised a quantitative assay for this morphological response, using the subline Kc-H. The assay was used to characterize the conditions required for the response. A halfmaximal response is elicited by approximately 10^–8M 20-hydroxyecdysone; the response is saturated by 10^–7M 20-hydroxyecdysone, which causes detectable elongation within a few hours, and a maximal response after 2–3 days. The response occurs substantially normally in the absence of serum, during growth in suspension, and in over-crowded cultures. It is not elicited by cyclic nucleotides, vertebrate growth factors, or a variety of other non-ecdysteroid reagents. Of 60 ecdysteroid compounds tested, only those which were active in other insect test systems elicited the response, and the concentrations required were approximately proportional to the concentrations active in other in vitro systems. We conclude that the response of Kc cells to 20-hydroxyecdysone retains basic features of the ecdysteroid response of intact tissues and therefore that Kc cells are a useful model system for studying ecdysteroid action.     

Characterization and partial purification of the Drosophila Kc cell ecdysteroid receptor

The molting hormones of insects, the ecdysteroids, are steroids whose action is mediated by an intracellular receptor. The Kc cell line of Drosophila melanogaster possesses ecdysteroid receptors and exhibits characteristic, receptor-dependent morphological and biochemical responses to the application of ecdysteroids. This paper describes the interaction of muristerone A (2 beta, 3 beta, 5 beta, 11 alpha, 14 alpha(20R,22R)- heptahydroxycholest-7-en-6-one), a phytoecdysteroid, with the Kc cell ecdysteroid receptor. Muristerone A-receptor complexes are not as sensitive to dissociation in high salt buffers as other ecdysteroid-receptor complexes we have examined. This has enabled us to use [3H]muristerone A to follow the Kc cell ecdysteroid receptor during heparin-agarose, DNA-cellulose, and hydroxylapatite chromatography, as well as gel filtration and ion exchange high pressure liquid chromatography. The Drosophila Kc cell ecdysteroid receptor has a Stokes radius of 4.6 nm, a frictional coefficient of 1.4, and a molecular weight of 120,000. A procedure is presented that results in a 750-fold enrichment of the receptor.     

An ecdysteroid-induced alteration in the cell cycle of cultured Drosophila cells

The addition of physiological concentrations of the arthropod molting hormone 20-hydroxyecdysone results in the cessation of cell division in the Kc cell line of Drosophila melanogaster. Fluorometric mononitoring of the cell cycle reveals that treatment of the cells with hormone for 12 hr causes a G2 arrest. The dose-response curves are in agreement with those obtained for other hormonal effects in both the Kc line and the intact animal. In the continual presence of hormone, cells remain G2-arrested for approximately 100 hr, resuming division by 120 hr. Cells which have responded once to ecdysteroids and subsequently reentered the cell cycle are insensitive to hormonal restimulation. This lack of response has been correlated with, and is probably due to, the loss of ecdysteroid receptors in stimulated cells.     

Identification of a putative azadirachtin-binding complex from Drosophila Kc167 cells

Cell-proliferation in Drosophila Kc167 cells was inhibited by 50% when cell cultures contained 1.7 x 10(-7) M azadirachtin for 48 h (a tertranortriterpenoid from the neem tree Azadirachta indica). Drosophila Kc167 cells exhibited direct nuclear damage within 6-h exposure to azadirachtin (5 x 10(-7) M and above) or within 24 h when lower concentrations were used (1 x 10(-9) M). Fractionation of an extract of Drosophila Kc167 cells combined with ligand overlay technique resulted in the identification of a putative azadirachtin binding complex. Identification of the members of this complex by Peptide Mass Fingerprinting (PMF) and N-terminal sequencing identified heat shock protein 60 (hsp60) as one of its components.     

Stereoselective synthesis of (22R)- and (22S)-castasterone/ponasterone A hybrid compounds and evaluation of their molting hormone activity

Two stereoisomers of a castasterone/ponasterone A hybrid compound, the (20R,22R) and (20R,22S)-isomers of 2alpha,3alpha,20,22-tetrahydroxy-5alpha-cholestan-6-one, were synthesized stereoselectively and their binding activity to the ecdysteroid receptor was determined. From the concentration-response curve for the inhibition of the incorporation of tritiated ponasterone A into ecdysteroid receptor containing insect cells, the concentration (IC50) required to inhibit 50% of the incorporation of radioactivity into cells was evaluated. The IC50 values of the (22R)- and (22S)-isomers were determined to be 0.30 and 38.9 microM against Kc cells, respectively, indicating that the (22R)-isomer is about 100 times more potent than the corresponding (22S)-isomer. IC50 values of these compounds against lepidopteran Sf-9 cells were determined to be 0.36 and 12.9 microM, respectively. The molting hormonal effect was examined in a Chilo suppressalis integument system and the 50% effective concentration for the stimulation of N-acetylglucosamine incorporation into the cultured integument was determined to be 2.7 microM for the (22R)-isomer, while the (22S)-isomer was inactive. On the other hand, both isomers did not show brassinolide-like activity in the rice lamina inclination assay.     

Two novel LEM-domain proteins are splice products of the annotated Drosophila melanogaster gene CG9424 (Bocksbeutel)

The LEM motif is a sequence of 40-50 amino acids that has been identified in a number of non-related proteins of the inner nuclear membrane including the lamina-associated polypeptides 2 (LAP2), emerin, MAN1 and the Drosophila protein otefin. This evolutionary conserved sequence motif can mediate via the interaction with the small protein BAF the binding of LEM-domain proteins to DNA. Taking advantage of its sequenced genome we analyzed whether Drosophila possesses beside otefin additional genes coding for proteins with a LEM motif. A putative candidate gene was the annotated gene CG9424 which we named Bocksbeutel. Of all putative Drosophila LEM-domain proteins, otefin and Bocksbeutel exhibited the highest similarity in the LEM motif (53% identical amino acids). The Bocksbeutel gene can code for two isoforms of 399 and 351 amino acids that are produced by alternative splicing. In the alpha-isoform a transmembrane domain is localized close to the carboxyterminus. This segment is absent in the shorter beta-isoform. By RT-PCR we could show that in the embryo the mRNA coding for the alpha-isoform and in significantly lower amounts the mRNA coding for the beta-isoform are expressed. When expressed in transfected cells as GFP fusion proteins, the beta-isoform is localized predominantly in the nucleoplasm and the alpha-isoform is targeted to the nuclear envelope, indicating that Bocksbeutel-alpha is localized in the inner nuclear membrane. Bocksbeutel-alpha is the predominant isoform expressed in cells, larvae, and flies. Indirect immunofluorescence with Bocksbeutel-specific antibodies on tissues and cultured cells revealed that Bocksbeutel proteins are localized in the nuclear envelope and in the cytoplasm. By RNA interference we have down-regulated the expression of Bocksbeutel, BAF, otefin, and lamin DmO in Drosophila Kc167 cells. The down-regulation of Bocksbeutel and otefin had no influence on the viability of Kc167 cells and the intracellular localization of all other nuclear and nuclear envelope proteins analyzed. In contrast, when lamin DmO was reduced by RNAi the distribution of Bocksbeutel and otefin in the nuclear envelope of Kc167 cells was significantly altered. We conclude that the two LEM-domain proteins Bocksbeutel and otefin are no limiting components for the maintenance of the nuclear architecture in cultured Drosophila cells at interphase.     

Ecdysteroid binding sites in gastrolith forming tissue and stomach during the molting cycle of crayfish Procambarus clarkii.

The distribution of ecdysteroid binding sites in the stomach and gastrolith disc tissue of crayfish (Procambarus clarkii) was examined in relation to the molting stage by thaw-mount autoradiography. The radiolabeled hormone analogue ponasterone A (25-deoxy-20-hydroxyecdysone) was used. Ecdysteroid binding sites were demonstrated only in certain molting stages, the small gastrolith period and the aftermolt stage. In gastrolith epithelium, ponasterone A binding sites first appeared in the cytoplasm, and then in the nuclei and cytoplasm. In the stomach epithelium, many nuclear binding sites were detectable during the period of gastrolith secretion. These periodical changes in specific ponasterone A binding when correlated with the molting stages clearly show that ecdysteroids may function as an initiator for gastrolith formation and reabsorption. The findings also suggest that ecdysteroids control calcium transport in the stomach epithelium. The time-related and functional differences of cytoplasmic and nuclear concentration of ecdysteroid receptors indicate the presence of cytoplasmic and nuclear receptors associated with specific actions.     

Inhibition of [3H]ponasterone A binding by ecdysone agonists in the intact Kc cell line.

Inhibition of the binding of [3H]ponasterone A ([3H]PoA) by ecdysone agonists including diacylhydrazines such as RH-5849, tebufenozide (RH-5992) and methoxyfenozide (RH-2485) was examined in intact Drosophila Kc cells. The reciprocal logarithm of the concentration at which there is 50% inhibition of [3H]PoA binding, pIC(50) (M), was determined as the binding activity for all compounds from each concentration-response curve. The order of the activity was PoA>20-hydroxyecdysone>cyasterone>inokosterone>or=makisterone A>methoxyfenozide>or=tebufenozide>ecdysone>RH-5849. The ranking of steroidal ecdysone analogs is consistent with that obtained against Spodoptera Sf-9 cells. Furthermore, in terms of pIC(50), all binding activity for ecdysone analogs, except ecdysone, estimated in the Kc cell line system was significantly higher than that for the Sf-9 cell line system. However, the activity of ecdysone was comparable between Kc and Sf-9 cells. The activity of diacylhydrazine analogs against Kc cells was significantly low compared with that against Sf-9 cells. The potency of methoxyfenozide was 1/200 that of PoA, which showed the highest activity in the Kc cell line system among all compounds tested. The activity of tebufenozide analogs having an n-pentyl or n-hexyl group instead of a 4-ethylphenyl group was similar to that of RH-5849.     

Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors

The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.     

Ecdysteroid binding sites in gastrolith forming tissue and stomach during the molting cycle of crayfishProcambarus clarkii

Summary The distribution of ecdysteroid binding sites in the stomach and gastrolith disc tissue of cryafish (Procambarus clarkii) was examined in relation to the molting stage by thaw-mount autoradiography. The radiolabeled hormone analogue ponasterone A (25-deoxy-20-hydroxyecdysone) was used. Ecdysteroid binding sites were demonstrated only in certain molting stages, the small gastrolith period and the aftermolt stage. In gastrolith epithelium, ponasterone A binding sites first appeared in the cytoplasm, and then in the nuclei and cytoplasm. In the stomach epithelium, many nuclear binding sites were detectable during the period of gastrolith secretion. These periodical changes in specific ponasterone A binding when correlated with the molting stages clearly show that ecdysteroids may function as an initiator for gastrolith formation and reabsorption. The findings also suggest that ecdysteroids control calcium transport in the stomach epithelium. The time-related and functional differences of cytoplasmic and nuclear concentration of ecdysteroid receptors indicate the presence of cytoplasmic and nuclear receptors associated with specific actions.     

Isopentenoid synthesis in embryonic Drosophila cells: prenylated protein profile and prenyl group usage.

It has been established that vertebrates and yeasts modified a unique subset of polypeptides with farnesyl and geranylgeranyl residues. This observation has been extended to Drosophila Kc cells. [3H]Mevalonate was incorporated into 54 Kc cell peptides (18-92 kDa). As reported for mammalian cells, most of the labeled peptides had molecular weights between 21 and 27 kDa. C18 radio-HPLC tryptic digest profiles for delipidized, [3H]mevalonate-labeled (a) insect (Drosophila and Spodoptera frugiperda) and mammalian (Chinese hamster ovary met 18-2b) cells, (b) Kc cell nuclear lamin, and (c) a 23.5-kDa purified Kc cell GTP-binding protein were compared and analyzed. [35S]Cysteine-labeled Kc cells yielded a tryptic digest radio-HPLC profile which was congruent with that for [3H]mevalonate-labeled cells. A significant fraction (30-33%) of the doubly labeled tryptic peptides were eluted with greater than or equal to 93% acetonitrile. Kc cell nuclear lamin tryptic digests yielded a single 3H-labeled product which migrated as S-farnesylcysteine. The Kc cell 23.5-kDa GTP-binding protein's 3H-labeled oligopeptide(s)/amino acid(s) was geranylgeranylated and its tryptic digest profile was representative of prenylated proteins whose oligopeptides eluted with greater than or equal to 93% acetonitrile. Moreover, the 3H-labeled oligopeptide/amino acid profiles plus prenyl group patterns for [3H]mevalonate-labeled Kc and mammalian cell total extracts were similar. Collectively, these observations supported a prenylated protein spectrum and prenyl group usage as highly conserved eukaryotic cellular characteristics.     

hormone nuclear receptor (EcR) exhibits circadian cycling in certain tissues, but not others, during development in Rhodnius prolixus (Hemiptera)

The insect moulting hormones, viz. the ecdysteroids, regulate gene expression during development by binding to an intracellular protein, the ecdysteroid receptor (EcR). In the insect Rhodnius prolixus, circulating levels of ecdysteroids exhibit a robust circadian rhythm. This paper demonstrates associated circadian rhythms in the abundance and distribution of EcR in several major target tissues of ecdysteroids, but not in others. Quantitative analysis of immunofluorescence images obtained by confocal laser-scanning microscopy following the use of anti-EcR has revealed a marked daily rhythm in the nuclear abundance of EcR in cells of the abdominal epidermis, brain, fat body, oenocytes and rectal epithelium of Rhodnius. This EcR rhythm is synchronous with the rhythm of circulating hormone levels. It free-runs in continuous darkness for several cycles, showing that EcR nuclear abundance is under circadian control. Circadian control of a nuclear receptor has not been shown previously in any animal. We infer that the above cell types detect and respond to the temporal signals in the rhythmic ecdysteroid titre. In several cell types, the rhythm in cytoplasmic EcR peaks several hours prior to the EcR peak in the nucleus each day, thereby implying a daily migration of EcR from the cytoplasm to the nucleus. This finding shows that EcR is not a constitutive nuclear receptor, as has previously been assumed. In the brain, rhythmic nuclear EcR has been found in peptidergic neurosecretory cells, indicating a potential pathway for feedback regulation of the neuroendocrine system by ecdysteroids, and also in regions containing circadian clock neurons, suggesting that the circadian timing system in the brain is also sensitive to rhythmic ecdysteroid signals. This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.     

Evaluation of hydrogen bonds of ecdysteroids in the ligand–receptor interactions using a protein modeling system

The insect molting hormone, 20-hydroxyecdysone (20E) and its analogs (ecdysteroids) specifically bind to the ecdysone receptor. Previously, we synthesized various ecdysteroids containing the side chain moiety of ponasterone A (PonA), and measured the binding activity against Drosophila Kc cells to study the structure–activity relationship. Here we quantitatively analyzed the structure–activity relationship for the ligand binding of ecdysteroids including 20E and PonA. Since the hydrogen bonding (HB) is one of the important physicochemical properties for ligand binding to the ecdysteroid receptor, the number of possible HBs between the ligand molecule and the receptor was manually counted in the modeled ligand–receptor complex for all compounds. The construction of the ligand–receptor model was executed by the full-automatic modeling system (FAMS) in which calculation was done by simulated annealing. The binding potency of 15 ecdysteroids to Kc-cells were linearly correlated (r² = 0.63) with the number of HBs which are observed between ligand and receptor molecule. Contribution of steric and electrostatic effects on the ligand–receptor binding was also examined using a three-dimensional quantitative structure–activity relationship (3-D QSAR), comparative molecular field analysis (CoMFA).     

Syndecan-dependent binding of Drosophila hemocytes to laminin alpha3/5 chain LG4-5 modules: potential role in sessile hemocyte islets formation

Heparin-column chromatography and elastase-digestion of medium from hemocyte Kc167 gave Drosophila laminin alpha3/5betagamma trimer, alpha3/5LG2-3 and alpha3/5LG4-5 modules with eluting NaCl concentrations of 450, 280 and 450 mM, respectively. Kc167 cells bound dish surface with alpha3/5betagamma trimer or alpha3/5LG4-5, but not with alpha3/5LG2-3 modules. Cell binding was counteracted by treating with heparin or heparan sulfate. RNA interference of syndecan in Kc167 cells impaired the binding, but that of dally or dally-like did not. Green fluorescent protein-expressing hemocytes also bound surface with alpha3/5betagamma trimer or alpha3/5LG4-5 module. Thus, syndecan-dependent binding of hemocytes to laminin may have a potential role in sessile hemocytes islets formation in T2-A8 segments of Drosophila.     

The Drosophila orphan nuclear receptor DHR38 mediates an atypical ecdysteroid signaling pathway

Ecdysteroid pulses trigger the major developmental transitions during the Drosophila life cycle. These hormonal responses are thought to be mediated by the ecdysteroid receptor (EcR) and its heterodimeric partner Ultraspiracle (USP). We provide evidence for a second ecdysteroid signaling pathway mediated by DHR38, the Drosophila ortholog of the mammalian NGFI-B subfamily of orphan nuclear receptors. DHR38 also heterodimerizes with USP, and this complex responds to a distinct class of ecdysteroids in a manner that is independent of EcR. This response is unusual in that it does not involve direct binding of ecdysteroids to either DHR38 or USP. X-ray crystallographic analysis of DHR38 reveals the absence of both a classic ligand binding pocket and coactivator binding site, features that seem to be common to all NGFI-B subfamily members. Taken together, these data reveal the existence of a separate structural class of nuclear receptors that is conserved from fly to humans.     

Developmental profiles of ecdysteroids, ecdysteroid receptor mRNAs and DNA binding properties of ecdysteroid receptors in the Ixodid tick Amblyomma americanum (L.)

Total body ecdysteroid titers were determined at specific stages during the larval and nymphal life of Amblyomma americanum (L.). One ecdysteroid peak was observed following the completion of larval apolysis. However, two distinct ecdysteroid peaks occurred at a comparable stage in the nymphal molting cycle. The first occurred following apolysis and the second peak occurred at about the time of ecdysis. When whole body profiles of EcR and RXR mRNAs were examined during the molting cycle using RT-PCR, the expression of both AamEcR and AamRXR mRNAs was shown to be correlated with the ecdysteroid titer. Using an electrophoretic gel mobility shift assay, it was demonstrated that AamEcR*AamRXR1, but not AamEcR*AamRXR2, exhibits broad DNA binding specificity, forming complexes with a variety of synthetic direct repeat and palindromic nuclear response elements with the half-site consensus AGGTCA. These data suggest that functional differences may exist between the AamRXR1 and AamRXR2 proteins.     

Scaffold attachment of DNA loops in metaphase chromosomes

We have examined the higher-order loop organization of DNA in interphase nuclei and metaphase chromosomes from Drosophila Kc cells, and we detect no changes in the distribution of scaffold-attached regions (SARs) between these two phases of the cell cycle. The SARs, previously defined from experiments with interphase nuclei, not only are bound to the metaphase scaffold when endogenous DNA is probed but also rebind specifically to metaphase scaffolds when added exogenously as cloned, end-labeled fragments. Since metaphase scaffolds have a simpler protein pattern than interphase nuclear scaffolds, and both have a similar binding capacity, it appears that the population of proteins required for the specific scaffold-DNA interaction is limited to those found in metaphase scaffolds. Surprisingly, metaphase scaffolds isolated from Drosophila Kc cells contain both the lamin protein and a pore-complex protein, glycoprotein (gp) 188. To study whether lamin contributes to the SAR-scaffold interaction, we have carried out comparative binding studies with scaffolds from HeLa metaphase chromosomes, which are free of lamina, and from HeLa interphase nuclei. All Drosophila SAR fragments tested bind with excellent specificity to HeLa interphase scaffolds, whereas a subset of them bind to HeLa metaphase scaffolds. The maintenance of the scaffold-DNA interaction in metaphase indicates that lamin proteins are not involved in the attachment site for at least a subset of Drosophila SARs. This evolutionary and cell-cycle conservation of scaffold binding sites is consistent with a fundamental role for these fragments in the organization of the genome into looped domains.     

Action of 24-epibrassinolide on a cell line of the beet armyworm, Spodoptera exigua

The Spodoptera exigua cell line Se4 is sensitive for ecdysteroid activity stimulated by the insect molting hormone, 20-hydroxyecdysone (20E), showing a cease in cell proliferation (with 50% inhibition around 1 microM) and characteristic cell morphology changes with aggregation and formation of long filamentous cytoplasmic extensions. The bisacylhydrazine tebufenozide also triggered such typical cellular effects in Se4, and in addition, it showed an affinity for binding in competition with 3H-ponasterone A (PoA) that was similar to 20E (with 50% competition around 1 microM), confirming that such non-ecdysteroids display an ecdysteroid agonist activity. In contrast, when Se4 cells were incubated with the native plant hormone 24-epibrassinolide (24BR), none of the effects triggered by 20E were observed. Hence, a competition binding experiment with 3H-PoA demonstrated no affinity of 24BR for binding to the ecdysteroid receptor in the Se4 cell line. In another series of experiments, the Se4 cell line was tested in sensitivity response to increased acetylcholinesterase (AchE) activity after treatment with ecdysteroid active compounds. The AchE activity measured in the cell line is discussed in relation to inhibition by eserine. The obtained results suggest that 24BR exerted no ecdysteroid activity.