Characterization of ecdysteroid receptors in cytosol and naive nuclear preparations of Drosophila Kc cells

Significant ecdysteroid binding activity can be demonstrated in nuclear extracts obtained from hormonally naive Drosophila Kc cells. The kinetic and physical characteristics of this nuclear binding are presented and compared with those exhibited by a high speed cytosol preparation of Kc cells. Examination of the effect of in vivo ecdysteroid exposure on the number of nuclear binding sites revealed that the quantity of nuclear receptors was not detectably altered. In addition, an effective synthesis of the biologically active ecdysteroid radioligand, [3H]ponasterone A, is described.     

Development of a cell-based assay for ecdysteroid quantification using an early ecdysteroid-inducible gene promoter

Ecdysteroids, primarily 20-hydroxyecdysone (20E) and ecdysone (E), are steroid hormones that regulate various developmental and physiological processes in insects. Commonly, immunoassays are used to quantify ecdysteroid titers of insects. However, the antibodies used in these assays react not only with 20E and E but often also with their inactive reserves and metabolites, and thus require purification before they can be quantified precisely. Here, we developed a simple cell-based method to quantify only the hormonally active ecdysteroids using newly established cells harboring the firefly luciferase gene under the control of the ecdysteroid-inducible promoter of the E75A gene of the silkworm Bombyx mori L. These cells also constitutively expressed the Renilla luciferase gene using the baculovirus ie2 promoter for internal reference. This cell-based method detected hormonally active ecdysteroids with significantly higher sensitivity than their inactive metabolites. Hemolymph ecdysteroid titers, determined using a dual luciferase assay after exposing these cells to crude extracts of B. mori larval and pupal hemolymph, agreed well with the sum of the 20E and E titers, which were quantified individually using a radioimmunoassay after they had been separated by HPLC. Thus, this method is very useful for quantifying the ecdysteroid titers of insects, particularly when the samples contain large amounts of ecdysteroid reserves and metabolites.     

Dual FRET assay for detecting receptor protein interaction with DNA

We present here a new assay that is based on the idea of the molecular beacon. This assay makes it possible to investigate two proteins interacting with DNA at two binding sites that are close to each other. The effectiveness of the test depends on the exclusive binding of three DNA fragments in the presence of two proteins, and the monitoring of the process depends upon observing the quenching of two independent fluorescence donors. As a model we used the components of the heterodimeric ecdysteroid receptor proteins ultraspiracle (Usp) and ecdysone receptor (EcR) from Drosophila melanogaster and a response element from the promoter of the hsp27 gene. The response element consists of two binding sites (half-sites) for the DNA binding domains (DBDs). We have shown that protein–protein interactions mediate cooperative binding of the ecdysteroid receptor DBDs to a hsp27_pal response element. The analysis of the microscopic dissociation constants obtained with the DMB led to the conclusion that there was increased affinity of UspDBD to the 5′ half-site in the presence of EcRDBD when the 3′ half-site was occupied, and increased affinity of EcRDBD to the 3′ half-site when the 5′ half-site was occupied.     

Design and synthesis of N-alkyl-N′-substituted 2,4-dioxo-3,4-dihydropyrimidin-1-diacylhydrazine derivatives as ecdysone receptor agonist

Based on the discovery of thymine as an ecdysteroid agonist, a series of 1,4-disubstituted diacylhydrazine derivatives with a thymine moiety were designed and synthesized. The activities of these compounds against Spodoptera litura (Fabricius) were evaluated by the insect immersion method. Results showed that compound 2h with an N-cyclohexylmethyl substituent exhibits the most potent agonist activity with a median lethal concentration of 23.21 μg/mL. This compound also caused malformation of molting larvae and adults. Compound 2h was further demonstrated as an ecdysteroid agonist by reporter gene assay on the Spodoptera frugiperda cell line (Sf9 cells). A molecular docking study indicated that hydrophobic interactions and the formation of hydrogen bonds between the compounds and the ecdysone receptor play critical roles in promoting the binding affinity of the compound. The structure of compound 2h may serve as a favorable template for the development of new ecdysteroid agonists with a pyrimidinedione moiety.     

The effect of juvenile hormone on prothoracic gland function during the larval-pupal development of Manduca sexta: An in situ and in vitro analysis

The application of juvenile hormone I or ZR 512 to neck-ligated, day-5 fifth instar (V₅) larvae reduced the time to pupation in a dose-dependent manner when compared to neck-ligated controls treated with methyl epoxy stearate. Haemolymph ecdysteroid titres determined by radioimmunoassay (RIA) reflected the ability of juvenile hormone I and ZR 512 to stimulate larval-pupal development, i.e. the ecdysteroid titres were similar to those of normally developing larvae although the ecdysteroid peak elicited by ZR 512 lagged that in the normal titre by 1 day, while that elicited by juvenile hormone I lagged the ecdysteroid peak in normal larvae by 2 days. Neck-ligated V₅ larvae that were untreated ultimately pupated and the haemolymph ecdysteroid peak eliciting pupation in these animals was 7 μg/ml haemolymph, almost double that of normal animals and ZR 512- and juvenile hormone I-treated, ligated larvae. The data indicated that juvenile hormone I does stimulate the prothoracic glands but to determine whether this stimulation was direct or indirect, an in vitro approach was taken. Prothoracic glands from V₅, V₆ and V₇ larvae were incubated in vitro under conditions in which they could be stimulated by prothoracicotropic hormone, and were exposed to concentration of free juvenile hormones I, II, III or ZR 512 ranging from 10^?5M to 10^?10M. In no case were the prothoracic glands stimulated in a dose-dependent manner that would be indicative of hormone activation. Similar results were obtained when juvenile hormone bound to binding protein was incubated with the prothoracic glands. Studies with the acids of the three juvenile hormone homologues revealed them to be ineffective in activating prothoracic glands, although juvenile hormone III acid does appear to inhibit the synthesis of ecdysone by day-0 pupal prothoracic glands. The significance of the latter effect is unknown. It is concluded from these data that juvenile hormone can, indeed, activate late larval prothoracic glands in situ, but does so indirectly.     

Suppression of the ecdysteroid-triggered growth arrest by a novel Drosophila membrane steroid binding protein

Ecdysteroid is a crucial steroid hormone in insects, especially during metamorphosis. Here, we show that the Drosophila membrane steroid binding protein (Dm_MSBP) is a novel structural homolog of the vertebrate membrane-bound receptor component for progesterone. Dm_MSBP exhibited binding affinity to ecdysone when expressed on the cell surface of Drosophila S2 cells. In S2 cells, the stable overexpression of Dm_MSBP suppressed the growth arrest triggered by 20-hydroxyecdysone and prevented the temporal activation of extracellular signal-regulated kinase proteins. These results suggest that Dm_MSBP is a membranous suppressor to ecdysteroid and blocks the signaling by binding it in extracellular fluid.     

Phytoecdysteroids in seeds of Lloydia serotina (Liliaceae)

Seeds of a number of species in the Liliaceae (sensu Brummitt, 1992) were examined for the presence of ecdysteroid agonist and antagonist activities. No species were antagonistic to 20-hydroxyecdysone action on the ecdysteroid-responsive Drosophila melanogaster B_II cell line and only one extract, that of Lloydia serotina, was agonistic. This activity is attributable to the presence of phytoecdysteroids as detected by ecdysteroid-specific radioimmunoassay and the agonist version of the B_II bioassay. HPLC in conjunction with radioimmunoassay and bioassay have been used to determine the ecdysteroid profile. The major ecdysteroids present are identified as 20-hydroxyecdysone and polypodine B (5β,20-dihydroxyecdysone).     

Ecdysteroid regulation of ovarian growth and oocyte maturation in the red flour beetle,Tribolium castaneum

Previous studies from our laboratory showed the involvement of juvenile hormone (JH) and ecdysteroid signaling in the regulation of female reproduction in the red flour beetle, Tribolium castaneum. JH regulates vitellogenin (Vg) synthesis in the fat body but the role of ecdysteroid signaling is not known. Here, we report on ecdysteroid regulation of ovarian growth and oocyte maturation. Microarray analysis of RNA isolated from ovaries showed the up-regulation of several genes coding for proteins involved in ecdysteroid signaling on the 4th day after female adult eclosion. The functional analyses of genes coding for proteins involved in ecdysteroid and JH signaling pathways by RNA interference (RNAi) revealed that ecdysteroids but not JH regulate ovarian growth and primary oocyte maturation. Ultrastructural studies showed the temporal sequences of key events in oogenesis including the development of primary oocytes, the differentiation and development of follicle epithelial cells, and the formation of intercellular spaces to facilitate uptake of Vg protein. RNAi studies showed that ecdysone receptor (EcR) and ultraspiracle (USP) are required for the ovarian growth, primary oocyte maturation and the growth and migration of the follicle cells. These studies suggest important roles for ecdysteroids in the regulation of oocyte maturation in the beetle ovaries.     

Makisterone A: The major ecdysteroid from the pupa of the honey bee,Apis mellifera

Makisterone $\text{A}$, a 28-carbon moulting hormone, has been identified as the major free pupal ecdysteroid in the honey bee, Apis mellifera. The pupal ecdysteroid was isolated and identified by normal and reversed-phase high performance liquid chromatography in conjunction with a radioimmune assay. The compound was further characterized physico-chemically by both mass spectrometry and nuclear magnetic resonance spectroscopy. No C₂₇ ecdysteroids (i.e. 20-hydroxyecdysone or ecdysone) were detected at this stage of development. This is the first isolation and identification of a 28-carbon ecdysteroid in an insect species from the order Hymenoptera. Utilization of dietary sterols by honey bees is also discussed.     

Luteinizing hormone stimulates the production of prostacyclin by isolated ovarian cell invitro

Granulosa cells isolated from mature Graafian follicles of swine produced significant quantities of immunoreactive 6-keto-PGF₁α under chemically defined conditions in vitro. Luteinizing hormone elicited a dose-dependent stimulation of 6-keto-PGF₁α accumulation, but follicle stimulating hormone, prolactin, L-epinephrine, estradiol-17B, or PGE₂ were devoid of effect. The time-dependent in vitro production of 6-keto-PGF₁α by ovarian cells was susceptible to inhibition by indomethacin, U-51506, cycloheximide, and actinomycin D. These observations implicate granulosa cells in the specific and hormonally regulated production of prostacyclin.     

Creation of ecdysone receptor chimeras in plants for controlled regulation of gene expression

Transformation with a chimeric receptor containing the glucocorticoid transactivation and DNA-binding domains fused to an ecdysteroid receptor ligand-binding domain permits ecdysone agonist-inducible gene expression in monocotyledonous plant cells. The inducible system is based on the specific activation of a chimeric receptor containing the ligand-binding domain of the Heliothis virescens ecdysteroid receptor and the inducer RH5992 (a 20-hydroxyecdysone agonist). RH5992 is an non-steroidal agrochemical with a high specificity for lepidopteran ecdysone receptors. Addition of RH5992 to transformed cells results in high levels of inducible expression in a ligand-specific manner, particularly when the effector receptor is coupled to the strong transactivator VP16. A chimeric construct containing the Drosophila ecdysone ligand-binding domain failed to activate reporter gene activity with RH5992, while activation was observed in the presence of muristeroneA. The system described provides the basis for an inducible gene expression system that is compatible with agricultural use. Received: 24 September 1998 / Accepted: 15 January 1999     

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic ‘eat-me' signals: cleavage and inhibition of phagocytosis by Lp-PLA2

Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H₂O₂ was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A₂ (Lp-PLA₂), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA₂ was blocked by a selective inhibitor of Lp-PLA₂, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic ‘eat-me'' signal that participates in the engulfment of cells undergoing intrinsic apoptosis.     

The hormonal regulation and interaction of concanavalin A and insulin binding in R3230AC mammary adenocarcinoma cells

To investigate the effects of concanavalin A on insulin binding to R323AC mammary carcinomas, initial experiments were performed to characterize binding of concanavalin A. Concanavalin A binding was found to be specific and saturable. Equilibrium binding experiments demonstrated that addition of low concentration of concanavalin A enhanced the binding of [³H]concanavalin A, suggestive of positively cooperative interactions. Binding of concanavalin A was responsive to hormonal alterations; tumor cells from diabetic rats showed enhanced binding of concanavalin A and insulin compared to cells from intact rats and administration of insulin to diabetic rats returned concanavalin A and insulin binding to levels seen in controls. Incubation of tumor cells with concanavalin A prior to addition of ¹²⁵I-labelled insulin resulted in a reduction of insulin-binding capacity; succinyl-concanavalin A did not affect binding of insulin. The percent inhibition of insulin binding by concanavalin A was highest at the lower insulin concentrations, providing a linearized Scatchard plot that yielded a calculated K_d value comparable to the low-affinity portion of the curvilinear Scatchard plot for insulin binding. The dissociation rate of bound insulin depended on receptor occupancy. Addition of concanavalin A after insulin binding reached equilibrium resulted in increased insulin binding hormone concentrations, decreased rates of dissociation of insulin and a loss of the correlation between receptor occupancy and dissociation rates. Concanavalin A alone demonstrated an insulin-like effect on glucose transport, which in these tumor cells represents a decrease in transport of 3-O-methylglucose. These suggest that binding of both concanavalin A and insulin to cells from this hormonally responsive neoplasm is under insulin regulation and demonstrates similar characteristics to those reported for a variety of normal cells. Furthermore, the interaction between concanavalin A and the cell membranes affects the affinity of the insulin receptor for insulin and appears to decrease the observed negative cooperativity.     

Identification of the endogenous apolar ecdysteroid conjugates present in newly-laid eggs of the house cricket (Acheta domesticus) as 22-long-chain fatty acyl esters of ecdysone

Newly-laid eggs of the house cricket Acheta domesticus contain significant amounts of apolar ecdysteroid conjugates, which can be hydrolysed by prolonged incubation with a mixture of Helix pomatia gut hydrolases. The ecdysteroid released on hydrolysis of the apolar conjugates has been purified and identified as ecdysone by co-chromatography on normal-phase and reversed-phase HPLC and by fast-atom bombardment mass spectrometry.Starting with only 22 g newly-laid eggs (containing 16 μg conjugated ecdysone), the ecdysone conjugates have been purified by open column chromatography and four successive HPLC purification steps to give essentially pure apolar conjugates with a yield of 57%. The conjugates are shown to be a mixture of ecdysone 22-fatty acyl esters by co-chromatography with authentic reference compounds and by fast-atom bombardment mass spectrometry. The fatty acyl composition of the conjugates is very similar to that produced by the ovaries of A. domesticus from [³H]ecdysone in vitro (Whiting and Dinan, Biochem. J.252, 95–103, 1988). The major fatty acyl esters are the 22-palmitate (C_16:0), 22-oleate (C_18:1) and 22-linoleate (C_18:2), with smaller amounts of the myristate (C_14:0), stearate (C_18:0) and arachidate (C_20:0) esters.This report constitutes the first identification from an insect source of endogenous ecdysteroid 22-fatty acyl esters, which have previously been identified in ticks and as metabolites of exogenous [³H]ecdysone in several arthropod species.     

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGFβ and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44^low, CD62L^high) and resting (CD25^-, CD69^-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.     

Differentiation of Human T Cells Alters Their Repertoire of G Protein α-Subunits

Because T cell differentiation leads to an expanded repertoire of chemokine receptors, a subgroup of G protein-coupled receptors, we hypothesized that the repertoire of G proteins might be altered in parallel. We analyzed the abundance of mRNA and/or protein of six G protein α-subunits in human CD4⁺ and CD8⁺ T cell subsets from blood. Although most G protein α-subunits were similarly expressed in all subsets, the abundance of Gα_o, a protein not previously described in hematopoietic cells, was much higher in memory versus naive cells. Consistent with these data, activation of naive CD4⁺ T cells in vitro significantly increased the abundance of Gα_o in cells stimulated under nonpolarizing or T_H17 (but not T_H1 or T_H2)-polarizing conditions. In functional studies, the use of a chimeric G protein α-subunit, Gα_qo5, demonstrated that chemokine receptors could couple to Gα_o-containing G proteins. We also found that Gα_i1, another α-subunit not described previously in leukocytes, was expressed in naive T cells but virtually absent from memory subsets. Corresponding to their patterns of expression, siRNA-mediated knockdown of Gα_o in memory (but not naive) and Gα_i1 in naive (but not memory) CD4⁺ T cells inhibited chemokine-dependent migration. Moreover, although even in Gα_o- and Gα_i1-expressing cells mRNAs of these α-subunits were much less abundant than Gα_i2 or Gα_i3, knockdown of any of these subunits impaired chemokine receptor-mediated migration similarly. Together, our data reveal a change in the repertoire of Gα_i/o subunits during T cell differentiation and suggest functional equivalence among Gα_i/o subunits irrespective of their relative abundance.     

Ecdysteroid promotes cell cycle progression in the Bombyx wing disc through activation of c-Myc

Developmental switching from growth to metamorphosis in imaginal primordia is an essential process of adult body planning in holometabolous insects. Although it is disciplined by a sequential action of the ecdysteroid, molecular mechanisms linking to cell proliferation are poorly understood. In the present study, we investigated the expression control of cell cycle–related genes by the ecdysteroid using the wing disc of the final-instar larvae of the silkworm, Bombyx mori. We found that the expression level of c-myc was remarkably elevated in the post-feeding cell proliferation phase, which coincided with a small increase in ecdysteroid titer. An in vitro wing disc culture showed that supplementation of the moderate level of the ecdysteroid upregulated c-myc expression within an hour and subsequently increased the expression of cell cycle core regulators, including A-, B-, D-, and E-type cyclin genes, Cdc25 and E2F1. We demonstrated that c-myc upregulation by the ecdysteroid was not inhibited in the presence of a protein synthesis inhibitor, suggesting a possibility that the ecdysteroid directly stimulates c-myc expression. Finally, results from the administration of a c-Myc inhibitor demonstrated that c-Myc plays an essential role in 20E-inducible cell proliferation. These findings suggested a novel pathway for ecdysteroid-inducible cell proliferation in insects, and it is likely to be conserved between insects and mammals in terms of steroid hormone regulation.