Qisheng Song

Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila.

Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Here we report that genetic ablation of the corpus allatum cells of the Drosophila ring gland (the JH source) resulted in JH deficiency, pupal lethality and precocious and enhanced programmed cell death (PCD) of the larval fat body. In the fat body of the JH-deficient animals, Dronc and Drice, two caspase genes that are crucial for PCD induced by the molting hormone 20-hydroxyecdysone (20E), were significantly upregulated. These results demonstrated that JH antagonizes 20E-induced PCD by restricting the mRNA levels of Dronc and Drice. The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of the 20E receptor EcR. Moreover, MET and GCE, the bHLH-PAS transcription factors involved in JH action, were shown to induce PCD by upregulating Dronc and Drice. In the Met- and gce-deficient animals, Dronc and Drice were downregulated, whereas in the Met-overexpression fat body, Dronc and Drice were significantly upregulated leading to precocious and enhanced PCD, and this upregulation could be suppressed by application of the JH agonist methoprene. For the first time, we demonstrate that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila.

Alterations in ultraspiracle (USP) content and phosphorylation state accompany feedback regulation of ecdysone synthesis in the insect prothoracic gland.

Insect molting and metamorphosis are elicited by a class of ecdysteroids, mainly 20-hydroxyecdysone (20E), the precursor of which is synthesized in the prothoracic gland. 20E acts via the ecdysone receptor (EcR) and its heterodimer partner ultraspiracle (USP). Analysis of the prothoracic gland of Manduca sexta revealed that the developmental expression and phosphorylation of a specific USP form, p47, is positively correlated with ecdysteroidogenesis and that 20E, but not ecdysone, is responsible for initiating the translational expression and phosphorylation of p47. The latter forms a functional complex with EcR and the ligand-complex interaction results in the down regulation of ecdysteroidogenesis and the inhibition of prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis. The composite data suggest that USP plays a key role in modulating PTTH-stimulated ecdysteroid biosynthesis through the selective expression and phosphorylation of the p47 USP isoform.

Multiple phosphorylation of ribosomal protein s6 and specific protein synthesis are required for prothoracicotropic hormone-stimulated ecdysteroid biosynthesis in the prothoracic glands of Manduca sexta

Prothoracicotropic hormone (PTTH)-stimulated protein phosphorylation leads to ecdysteroidogenesis (molting hormone biosynthesis) in the prothoracic glands of the tobacco hornworm, Manduca sexta. The phosphorylation of 34 and 50 kDa peptides (p34 and p50) paralleled the increase in ecdysteroidogenesis, and the dephosphorylation of p34 and p50 preceded a decrease in ecdysteroidogenesis. Inhibition by rapamycin of p34, but not p50, phosphorylation prevented PTTH-stimulated ecdysteroidogenesis in a dose-dependent manner, suggesting that p34 phosphorylation is requisite for PTTH-stimulated ecdysteroidogenesis. Two proteins whose synthesis was rapidly stimulated by PTTH were p50 and p70. The time-course of PTTH-stimulated synthesis of p50 paralleled that of p34 phosphorylation and that of ecdysteroidogenesis. Rapamycin inhibited PTTH-stimulated synthesis of p50 and p70, suggesting that specific protein synthesis is also required for PTTH-stimulated ecdysteroidogenesis, confirming the results of Rybczynski and Gilbert [(1994) Insect Biochem. Molec. Biol. 24, 175-189], and that p34 phosphorylation may regulate the downstream synthesis of p50 and p70, possible key regulatory proteins leading to ecdysteroidogenesis. Results from two-dimensional (2D)-PAGE analysis of the ribosomal proteins purified from prothoracic glands, demonstrated that p34 is indeed ribosomal S6, and is phosphorylated at up to five sites (P1-5) upon PTTH stimulation. The multiple phosphorylation of S6 was inhibited completely by rapamycin as shown in 2D gel maps, further confirming that p34 is ribosomal protein S6. Temporal analysis of PTTH-stimulated S6 phosphorylation by 2D-PAGE revealed that phosphorylation of S6 at the P1 site was temporally correlated with the initiation of ecdysteroidogenesis, and that multiple phosphorylation at all five sites (P1-5) was correlated with the maximal synthesis of ecdysteroids. Dephosphorylation of S6 was accompanied by a decrease in ecdysteroidogenesis. These data demonstrate that p34 is ribosomal protein S6 and that both the phosphorylation of S6 and specific protein synthesis are required for PTTH-stimulated ecdysteroidogenesis in the prothoracic gland.

Function of nuclear transport factor 2 and Ran in the 20E signal transduction pathway in the cotton bollworm, Helicoverpa armigera

BackgroundNuclear transport factor 2 and small GTPase Ran participate in the nucleo-cytoplasm transport of macromolecules, but their function in the 20-hydroxyecdysone (20E) signal transduction pathway are not well known.ResultsA 703 bp encoding Ntf2 and a 1233 bp encoding Ran full-length cDNAs were cloned from Helicoverpa armigera, and named Ha-Ntf2 and Ha-Ran, respectively. Northern blot and immunoblotting revealed that Ha-Ntf2 had an obviously higher expression levels in the head-thorax and integument of the metamorphically committed larvae. In contrast, the expression of Ha-Ran did not show obvious variation at various developmental stages in four tissues by immunoblotting analysis, except in the midgut, which showed increased expression from 5th-36 h (molting) to 6th-48 h. Both expressions of Ha-Ntf2 and Ha-Ran could be upregulated by 20E in vitro. Immunohistochemistry revealed that Ha-Ntf2 and Ha-Ran were primarily localized in the nucleus of various tissues. Protein binding assay and co-immunoprecipitation indicated that Ha-Ntf2 and Ha-Ran can combine with each other in vitro and in vivo. Knock down of Ha-Ntf2 or Ha-Ran by RNAi resulted in the suppression of other 20E regulated genes including EcR-B1, USP1, E75B, BR-CZ2, HHR3 and Ha-eIF5c. In addition, the knockdown of Ha-Ntf2 resulted in Ha-Ran being prevented in the cytoplasm. The nuclear location of the ecdysone receptor b1 (EcR-B1) was also blocked after the knockdown of Ha-Ntf2 and Ha-Ran.ConclusionThese evidences suggested that Ha-Ntf2 and Ha-Ran participated in the 20E signal transduction pathway by regulating the location of EcR-B1.

An Immunophilin is a Component of the Insect Ecdysone Receptor (EcR) Complex

The ecdysone receptor (EcR) complex has been identified in the prothoracic gland of Manduca sexta by specific immunoprecipitation and Western blot analyses, and includes EcR, ultraspiracle (USP) and FKBP46. The EcR complex binds ponasterone A in a dose-dependent manner with a Kd of 7.04 x 10(-9) M. Immunocytochemistry revealed that EcR, USP and FKBP46 were localized within the nucleus of the prothoracic gland cells, and suggested that the developmental expression patterns of EcR and USP changed in concert with the hemolymph ecdysteroid titer whereas that of FKBP46 did not. The composite results suggest that the hemolymph ecdysteroid titer, of which 20 hydroxyecdysone is the major component, modulates the expression of both EcR and USP in the prothoracic gland to achieve feedback regulation.

Prostaglandins A1 and E1 influence gene expression in an established insect cell line (BCIRL-HzAM1 cells)

Prostaglandins (PGs) and other eicosanoids exert important physiological actions in insects and other invertebrates, including influencing ion transport and mediating cellular immune defense functions. Although these actions are very well documented, we have no information on the mechanisms of PGs actions in insect cells. Here we report on the outcomes of experiments designed to test our hypothesis that PGs modulate gene expression in an insect cell line established from pupal ovarian tissue of the moth Helicoverpa zea (BCIRL-HzAM1 cells). We treated cells with either PGA(1) or PGE(1) for 12 or 24h then analyzed cell lysates by 2-D electrophoresis. Analysis of the gels by densitometry revealed substantial changes in protein expression in some of the protein spots we analyzed. These spots were processed for mass spectrometric analysis by MALDI TOF/TOF, which yielded in silico protein identities for all 34 spots. The apparent changes in three of the proteins were confirmed by semi-quantative PCR, showing that the changes in mRNA expression were reflected in changes in protein expression. The 34 proteins were sorted into six categories, protein actions, lipid metabolism, signal transduction, protection, cell functions and metabolism. The findings support the hypothesis that one mechanism of PG action in insect cells is the modulation of gene expression.

Expression of immune-response genes in lepidopteran host is suppressed by venom from an endoparasitoid, Pteromalus puparum

BackgroundThe relationships between parasitoids and their insect hosts have attracted attention at two levels. First, the basic biology of host-parasitoid interactions is of fundamental interest. Second, parasitoids are widely used as biological control agents in sustainable agricultural programs. Females of the gregarious endoparasitoid Pteromalus puparum (Hymenoptera: Pteromalidae) inject venom along with eggs into their hosts. P. puparum does not inject polydnaviruses during oviposition. For this reason, P. puparum and its pupal host, the small white butterfly Pieris rapae (Lepidoptera: Pieridae), comprise an excellent model system for studying the influence of an endoparasitoid venom on the biology of the pupal host. P. puparum venom suppresses the immunity of its host, although the suppressive mechanisms are not fully understood. In this study, we tested our hypothesis that P. puparum venom influences host gene expression in the two main immunity-conferring tissues, hemocytes and fat body.ResultsAt 1 h post-venom injection, we recorded significant decreases in transcript levels of 217 EST clones (revealing 113 genes identified in silico, including 62 unknown contigs) derived from forward subtractive libraries of host hemocytes and in transcript levels of 288 EST clones (221 genes identified in silico, including 123 unknown contigs) from libraries of host fat body. These genes are related to insect immune response, cytoskeleton, cell cycle and apoptosis, metabolism, transport, stress response and transcriptional and translational regulation. We verified the reliability of the suppression subtractive hybridization (SSH) data with semi-quantitative RT-PCR analysis of a set of randomly selected genes. This analysis showed that most of the selected genes were down-regulated after venom injection.ConclusionsOur findings support our hypothesis that P. puparum venom influences gene expression in host hemocytes and fat body. Specifically, the venom treatments led to reductions in expression of a large number of genes. Many of the down-regulated genes act in immunity, although others act in non-immune areas of host biology. We conclude that the actions of venom on host gene expression influence immunity as well as other aspects of host biology in ways that benefit the development and emergence of the next generation of parasitoids.

Control of ecdysteroidogenesis: Activation and inhibition of prothoracic gland activity

The ecdysteroid hormones, mainly 20-hydroxyecdysone (20E), play a pivotal role in insect development by controlling gene expression involved in molting and metamorphosis. In the model insectManduca sexta the production of ecdysteroids by the prothoracic gland is acutely controlled by a brain neurohormone, prothoracicotropic hormone (PTTH). PTTH initiates a cascade of events that progresses from the influx of Ca2+ and cAMP generation through phosphorylation of the ribosomal protein S6 and S6-dependent protein synthesis, and concludes with an increase in the synthesis and export of ecdysteroids from the gland. Recent studies indicate that S6 phosphorylation probably controls the steroidogenic effect of PTTH by gating the translation of selected mRNAs whose protein products are required for increased ecdysteroid synthesis. Inhibition of S6 phosphorylation prevents an increase in PTTH-stimulated protein synthesis and subsequent ecdysteroid synthesis. Two of the proteins whose translations are specifically stimulated by PTTH have been identified, one being a β tubulin and the other a heat shock protein 70 family member. Current data suggest that these two proteins could be involved in supporting microtubule-dependent protein synthesis and ecdysone receptor assembly and/or function. Recent data also indicate that the 20E produced by the prothoracic gland feeds back upon the gland by increasing expression and phosphorylation of a specific USP isoform that is a constituent of the functional ecdysone receptor. Changes in the concentration and composition of the ecdysone receptor complex of the prothoracic gland could modulate the glands potential for ecdysteroid synthesis (e.g. feedback inhibition) by controlling the levels of enzymes or other proteins in the ecdysteroid biosynthetic pathway.

Molecular Cloning, Developmental Expression, and Phosphorylation of Ribosomal Protein S6 in the Endocrine Gland Responsible for Insect Molting

Phosphorylation of ribosomal protein S6 is requisite for prothoracicotropic hormone (PTTH)-stimulated specific protein synthesis and subsequent ecdysteroidogenesis in the prothoracic glands of the tobacco hornworm, Manduca sexta To better understand the role of S6 in regulating ecdysteroidogenesis, S6 cDNA was isolated from a Manduca prothoracic gland cDNA library and sequenced. The deduced protein is comprised of 253 amino acids, has a molecular weight of 29,038, and contains four copies of a 10-amino acid motif defining potential DNA-binding sites. This Manduca S6 possesses a consensus recognition sequence for the p70s6k binding domain as well as six seryl residues at the carboxyl-terminal sequence of 17 amino acids. Phosphoamino acid analysis revealed that the phosphorylation of Manduca prothoracic gland S6 is limited exclusively to serine residues. Although alterations in the quantity of S6 mRNA throughout the last larval instar and early pupal-adult development were not well correlated with the hemolymph ecdysteroid titer, developmental expression and phosphorylation of S6 were temporally correlated with PTTH release and the hemolymph ecdysteroid titer. These data provide additional evidence that S6 phosphorylation is a critical element in the transduction pathway leading to PTTH-stimulated ecdysteroidogenesis.

Insect neuropeptide bursicon homodimers induce innate immune and stress genes during molting by activating the NF-κB transcription factor Relish.

Background Bursicon is a heterodimer neuropeptide composed of two cystine knot proteins, bursicon α (burs α) and bursicon β (burs β), that elicits cuticle tanning (melanization and sclerotization) through the Drosophila leucine-rich repeats-containing G protein-coupled receptor 2 (DLGR2). Recent studies show that both bursicon subunits also form homodimers. However, biological functions of the homodimers have remained unknown until now. Methodology/Principal Findings In this report, we show in Drosophila melanogaster that both bursicon homodimers induced expression of genes encoding antimicrobial peptides (AMPs) in neck-ligated adults following recombinant homodimer injection and in larvae fat body after incubation with recombinant homodimers. These AMP genes were also up-regulated in 24 h old unligated flies (when the endogenous bursicon level is low) after injection of recombinant homodimers. Up-regulation of AMP genes by the homodimers was accompanied by reduced bacterial populations in fly assay preparations. The induction of AMP expression is via activation of the NF-κB transcription factor Relish in the immune deficiency (Imd) pathway. The influence of bursicon homodimers on immune function does not appear to act through the heterodimer receptor DLGR2, i.e. novel receptors exist for the homodimers. Conclusions/Significance Our results reveal a mechanism of CNS-regulated prophylactic innate immunity during molting via induced expression of genes encoding AMPs and genes of the Turandot family. Turandot genes are also up-regulated by a broader range of extreme insults. From these data we infer that CNS-generated bursicon homodimers mediate innate prophylactic immunity to both stress and infection during the vulnerable molting cycle.