The RXR ortholog USP suppresses early metamorphic processes in Drosophila in the absence of ecdysteroids

The steroid hormone 20-hydroxyecdysone (20E) initiates metamorphosis in insects by signaling through the ecdysone receptor complex, a heterodimer of the ecdysone receptor (EcR) and ultraspiracle (USP). Analysis of usp mutant clones in the wing disc of Drosophila shows that in the absence of USP, early hormone responsive genes such as EcR, DHR3 and E75B fail to up-regulate in response to 20E, but other genes that are normally expressed later, such as (&bgr;)-Ftz-F1 and the Z1 isoform of the Broad-Complex (BRC-Z1), are expressed precociously. Sensory neuron formation and axonal outgrowth, two early metamorphic events, also occur prematurely. In vitro experiments with cultured wing discs showed that BRC-Z1 expression and early metamorphic development are rendered steroid-independent in the usp mutant clones. These results are consistent with a model in which these latter processes are induced by a signal arising during the middle of the last larval stage but suppressed by the unliganded EcR/USP complex. Our observations suggest that silencing by the unliganded EcR/USP receptor and the subsequent release of silencing by moderate steroid levels may play an important role in coordinating early phases of steroid driven development.     

Expression,subcellular localization and protein‑protein interaction of four isoforms of EcR/USP in the common cutworm

Ecdysone receptor (EcR) and ultraspiracle (USP) form heterodimers to mediate ecdysteroid signaling during molting and metamorphosis. Various EcR/USP heterodimers have been reported. However, it is unclear what kind of EcR/USP combination is adopted by lepidopteran insects during the larval?pupal metamorphosis and whether the EcR/USP heterodimer varies among different tissues. To address these questions, two isoforms of each EcR and USP were cloned from the common cutworm, their messenger RNA expression patterns were examined by real‐time quantitative polymerase chain reaction in different tissues during the larval–pupal metamorphosis and in the midgut in response to hormonal induction. Furthermore, their subcellular localization and protein?protein interaction were explored by transient expression and far‐western blotting, respectively. All the four genes were significantly up‐regulated in prepuae and/or pupae. The expression profiles of EcRB1 and USP1 were nearly identical to each other in the epidermis, fat body and midgut, and a similar situation also applied to EcRA and USP2. The three genes responded to 20‐hydroxyecdysone (20E) induction except for USP2, and USP1 could be up‐regulated by both 20E and juvenile hormone. The four proteins mainly localized in the nucleus and the nuclear localization was promoted by 20E. The protein?protein interaction between each EcR and USP was found in vitro. These results suggest that two types of EcR/USP heterodimer (EcRA/USP2 and EcRB1/USP1) may exist simultaneously in the common cutworm, and the latter should play more important roles during the larval?pupal metamorphosis. In addition, the types of EcR/USP heterodimer do not vary in the tissues which undergo histolysis and regeneration during metamorphosis.     

Heterodimerization of ecdysone receptor and ultraspiracle on symmetric and asymmetric response elements

Heterodimerization of nuclear receptors is facilitated by the interaction of two dimerization interfaces: one spanning the DNA-binding (C domain) region and the adjacent hinge (D domain) region, and the other in the ligand-binding (E domain) region. Ultraspiracle (USP) heterodimerizes with ecdysone receptor (EcR) and this complex participates in ecdysone signal transduction. The natural ecdysone response elements (EcREs) discovered so far are asymmetric elements composed of either imperfect palindromes or direct repeats. However, gel mobility shift assays have shown that both symmetric (perfect palindromes) and asymmetric (imperfect palindromes and direct repeats) elements can bind to the EcR/USP complex. Therefore, we analyzed EcR/USP domains involved in heterodimerization on different types of response elements (RE). Gel shift assays using full-length and truncated EcR and USP proteins showed that heterodimerization of these two proteins in the presence of asymmetric RE (DR4 and the natural EcRE hsp27) requires both dimerization interfaces present in CD and E domains of both proteins. In contrast, the dimerization interface present in the E domain of either EcR or USP was not essential for heterodimerization on symmetric RE such as PAL1 or IR1. We conclude that the use of heterodimerization interfaces present in CD and E domains of EcR/USP depends on the nature of response elements they bind to.     

Identification of 20-hydroxyecdysone-inducible genes from larval brain of the silkworm, Bombyx mori, and their expression analysis

The insect brain secretes prothoracicotropic hormone (PTTH), which stimulates the prothoracic gland to synthesize ecdysone. The active metabolite of ecdysone, 20-hydroxyecdysone (20E), works through ecdysone receptor (EcR) and ultraspiracle (USP) to initiate molting and metamorphosis by regulating downstream genes. Previously, we found that EcR was expressed in the PTTH-producing neurosecretory cells (PTPCs) in larval brain of the silkworm Bombyx mori, suggesting that PTPCs function as the master cells of development under the regulation of 20E. To gain a better understanding of the molecular mechanism of the 20E control of PTPCs, we performed a comprehensive screening of genes induced by 20E using DNA microarray with brains of day-2 fifth instar silkworm larvae. Forty-one genes showed greater than twofold changes caused by artificial application of 20E. A subsequent semiquantitative screening identified ten genes upregulated by 20E, four of which were novel or not previously identified as 20E-response genes. Developmental profiling determined that two genes, UP4 and UP5, were correlated with the endogenous ecdysteroid titer. Whole-mount in situ hybridization showed exclusive expression of these two genes in two pairs of cells in the larval brain in response to 20E-induction, suggesting that the cells are PTPCs. BLAST searches revealed that UP4 and UP5 are Bombyx homologs of vrille and tarsal-less, respectively. The present study identifies 20E-induced genes that may be involved in the ecdysone signal hierarchies underlying pupal-adult development and/or the 20E regulation of PTPCs.     

EcR expression in the prothoracicotropic hormone-producing neurosecretory cells of the Bombyx mori brain

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis through binding with a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and ultraspiracle (USP). Expression of the specific isoforms EcR-A and EcR-B1 governs steroid-induced responses in the developing cells of the silkworm Bombyx mori. Here, analysis of EcR-A and EcR-B1 expression during larval-pupal development showed that both genes were up-regulated by 20E in the B. mori brain. Whole-mount in situ hybridization and immunohistochemistry revealed that EcR-A and EcR-B1 mRNAs and proteins were exclusively located in two pairs of lateral neurosecretory cells in the larval brain known as the prothoracicotropic hormone (PTTH)- producing cells (PTPCs). In the pupal brain, EcR-A and EcR-B1 expression was detected in tritocerebral cells and optic lobe cells in addition to PTPCs. As PTTH controls ecdysone secretion by the prothoracic gland, these results indicate that 20E-responsive PTPCs are the master cells of insect metamorphosis.     

昆虫蜕皮激素受体及其类似物的杀虫机制研究进展

昆虫的蜕皮、变态和繁殖受到蜕皮激素的严格调控。蜕皮激素作用靶标由蜕皮激素受体（ecdysteroid receptor, EcR）和超气门蛋白（ultraspiracle protein, USP）组成,蜕皮激素与EcR/USP作用启动蜕皮级联反应过程。昆虫EcR具有种类或类群的特异性,研究其结构、功能和调控机理在开发环境友好型新药剂和基因调控开关等方面具有重要指导作用。该文介绍了昆虫EcR的结构和功能特点,蜕皮激素及其类似物与EcR/USP的分子作用方式,以及基于EcR/USP的新杀虫剂创制和基因调控开关设计等方面的重要进展。     

Ecdysone receptor and ultraspiracle proteins are tyrosine phosphorylated during adult development of silkmoths

20-hydroxy ecdysone (20E) is essential to promote adult development in diapausing silkmoth pupae. Increases in protein tyrosine/serine-phosphorylations observed soon after 20E administration supported the initial hypothesis that activation of receptor tyrosine kinase-ras-MAPK pathway could be responsible for the growth promoting effects of 20E. This report pertains to the high levels of protein tyrosine phosphorylations (PTP) that occurred later during the growth to differentiation transition because of its novelty and relevance to 20E dependence of adult development. Further analyses demonstrated that both ecdysone receptor (EcR) and ultraspiracle (USP), the two dimerizing partners of the functional ecdysone receptor, are tyrosine phosphorylated coincidental with high PTP. Enhanced PTP during growth to differentiation transition and concomitant tyrosine phosphorylation of EcR and USP was shown to occur in another silkmoth species pointing to the necessity of similar protein tyrosine phosphorylation pathways for adult development. Properly timed increases in tissue protein tyrosine kinase (PTK) activity could explain the enhancement of PTP in the wing epidermis of both the silkmoths. Thymidine incorporation measurements showed that cessation of DNA synthesis preceded the increase in PTK activity thus emphasizing a role for PTP in aspects of tissue physiology related to differentiative events rather than cell proliferation. Phosphatase and tyrosine kinase inhibitors (Tyrphostins) had minimal effects on adult wing development in vivo. However, the escape of the adult from the pupal case was blocked by a tyrphostin indicating the importance of PTKs in eclosion.     

Critical role of desolvation in the binding of 20-hydroxyecdysone to the ecdysone receptor

The insect steroid hormone 20-hydroxyecdysone (20E) binds to its cognate nuclear receptor composed of the ecdysone receptor (EcR) and Ultraspiracle (USP) and triggers the main developmental transitions, in particular molting and metamorphosis. We present the crystal structure of the ligand-binding domains of EcR/USP in complex with 20E at 2.4A resolution and compare it with published structures of EcR/USP bound to ponasterone A (ponA). ponA is essentially identical to 20E but lacks the 25-OH group of 20E. The structure of 20E-bound EcR indicates that an additional hydrogen bond is formed compared with the ponA-bound receptor, yet, paradoxically, ponA has a significantly higher affinity for EcR than 20E. Theoretical studies based on docking and free energy methods lead to a rationale for understanding the difference in binding affinities between 20E and ponA. Results of the calculations indicate that the favorable contribution from the extra H-bond made by 25-OH of 20E is counterbalanced by its larger desolvation cost compared with that of ponA. The contribution of 25-OH to the binding affinity is further compared with those of 20- and 22-OH groups. Ligands that lack the 20- or 22-OH group are indeed known to bind less favorably to EcR than 20E, an effect opposite to that observed for ponA. The results indicate that their respective contributions to receptor-ligand complex stability reside mostly in their different contributions to solvation/desolvation. Together, the data demonstrate the critical role of ligand desolvation in determining binding affinity, with general implications for the binding of hormones to their cognate nuclear receptors.     

Inducible expression and pharmacology of recombinant NMDA receptors,composed of rat NR1a/NR2B subunits

An ecdysone-inducible mammalian expression system was used to study expression of recombinant N-methyl-D-aspartate (NMDA) receptors. Human embryonic kidney (HEK) 293 cells expressing the regulatory vector pVgRXR (EcR 293 cells) were transfected with rat NR1a and NR2B cDNAs using the inducible vector pIND (Invitrogen). Inducible expression of the NR2B subunit in cell clone designated EcR/rNR1a2B was investigated using quantitative RT-PCR and flow cytometry based immunocytochemical methods. The mRNA level of the NR2B subunits in EcR/rNRa2B cells was dependent on the concentration of the ecdysone analogue inducing agent, muristerone A (MuA). Similarly, NR2B subunit protein expression was higher in cells pre-treated with the inducing agent. Functionally active NMDA receptors were also detected in EcR/rNR1a2B cells after MuA induction. In presence of the inducing factor, NMDA-evoked ion currents as well as increase in cytoplasmic calcium-concentrations were measured using whole-cell patch clamp and fluorometric calcium measuring techniques. The pharmacological profile of the expressed NMDA receptors was characterised by comparing the inhibitory activity of several NR2B subunit selective NMDA antagonists in EcR/rNR1a2B cells with that observed in primary cultures of rat cortical neurones. Whereas the efficacies of the NR2B subunit selective NMDA antagonists were similar in EcR/rNR1a2B cells and in neurones, their maximal inhibitory effects were significantly higher in cells expressing NR1a/NR2B recombinant receptors. This study demonstrates that recombinant NMDA receptors can be expressed in an inducible way in non-neuronal cell lines using the ecdysone-inducible mammalian expression system. Such cell lines can be suitable tools in high throughput functional screening for potential subtype selective modulators of the NMDA receptor.     

Comparative effects of a non-steroidal ecdysone agonist RH-5992 and 20-hydroxyecdysone in a lepidopteran cell line (IAL-PID2)

The non-steroidal ecdysone agonist, RH-5992, exhibits ecdysteroid activities in vivo as well as in vitro more effectively than 20-hydroxyecdysone (20E). Using the IAL-PID2 cells derived from imaginal wing discs of last larval instar of Plodia interpunctella, we investigated the action of RH-5992 in the control of cell growth. Its effects on the proliferative activity of IAL-PID2 cells, the induction level in G2/M arrest and on the expression rate of Plodia B cyclin (PcycB), ecdysone B1-isoform (PIEcR-B1) and Ultraspiracle-2 isoform (PIUSP-2) were examined. From these cellular and molecular assays, our results brought evidence that RH-5992, like 20E, induced an inhibition on cell proliferation by blocking IAL-PID2 cells in G2/M phase. Moreover, this G2/M arrest was preceded by a decrease in the expression level of PcycB and a high induction of PIEcR-B1, PIUSP-2 mRNAs. Dose-response experiments revealed that RH-5992 was even more potent than 20E. On these parameters, we therefore suggest that the differential observed in the expression level of USP and EcR by RH-5992 and 20E could contribute to the difference observed for the biological potency of these two compounds.     

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.