Upregulation of the expression of prodeath serine/threonine protein kinase for programmed cell death by steroid hormone 20-hydroxyecdysone

Serine/threonine protein kinases phosphorylate protein substrates to initiate further cellular events. Different serine/threonine protein kinases have varied functions despite their highly conserved homology. We propose prodeath-S/TK, a prodeath serine/threonine protein kinase from the lepidopteran insect Helicoverpa armigera, promotes programmed cell death (PCD) during metamorphosis. Prodeath-S/TK is expressed in various tissues with a high expression level during molting and metamorphosis by 20-hydroxyecdysone (20E) induction. Prodeath-S/TK is localized in the larval midgut during metamorphosis. Prodeath-S/TK knockdown by injecting dsRNA into larval hemocoel suppresses the 20E-induced metamorphosis and PCD, as well as downregulates a set of genes involved in the PCD and 20E signaling pathway. 20E upregulates prodeath-S/TK expression through its nuclear receptor EcR-B1 and USP1. Prodeath-S/TK overexpression in the epidermal cell line leads to PCD with DNA fragmentation and the activation of caspases 3 and 7. Prodeath-S/TK plays role in the cytoplasm. The N-terminal and C-terminal sequences of prodeath-S/TK determine its subcellular location. These data indicate that prodeath-S/TK participates in PCD by regulating gene expression in the 20E signaling pathway.     

Sumoylation modulates 20-hydroxyecdysone signaling by maintaining USP protein levels in Drosophila

The nuclear receptor complex for the insect steroid hormone, 20-hydroxyecdysone (20E), is a heterodimer of EcR and USP. It has been shown that Drosophila EcR and USP can be sumoylated in mammalian cells, but it is unknown whether EcR-USP sumoylation naturally occurs in Drosophila. In Drosophila cells, USP, but not EcR, was sumoylated by Smt3, the only Drosophila SUMO protein. The presence of EcR enhanced USP sumoylation, which is further enhanced by 20E treatment. In addition to the Lys20 sumoylation site, five potential acceptor lysine residues in USP were predicted and verified. Mutation of the USP sumoylation sites or reduction of smt3 expression by RNAi attenuated 20E-induced reporter activity. Moreover, in the salivary glands, reducing smt3 expression by RNAi decreased 20E-induced reporter activity, gene expression, and autolysosome formation. Importantly, at least partially, the smt3 RNAi-mediated reduction in 20E signaling resulted from decreased protein levels of USP. In conclusion, sumoylation modulates 20E signaling by maintaining USP protein levels in Drosophila.     

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.     

Identification of 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway

Background

20-hydroxyecdysone (20E) and its receptor complex ecdysone receptor (EcR) and ultraspiracle (USP) play a crucial role in controlling development, metamorphosis, reproduction and diapause. The ligand-receptor complex 20E-EcR/USP directly activates a small set of early-response genes and a much larger set of late-response genes. However, ecdysone-responsive genes have not been previously characterized in the context of insect chitin biosynthesis.

Principal Findings

Here, we show that injection-based RNA interference (RNAi) directed towards a common region of the two isoforms of SeEcR in a lepidopteron insect Spodoptera exigua was effective, with phenotypes including a high mortality prior to pupation and developmental defects. After gene specific RNAi, chitin contents in the cuticle of an abnormal larva significantly decreased. The expression levels of five genes in the chitin biosynthesis pathway, SeTre-1, SeG6PI, SeUAP, SeCHSA and SeCHSB, were significantly reduced, while there was no difference in the expression of SeTre-2 prior to 72 hr after injection of EcR dsRNA. Meanwhile, injection of 20E in vivo induced the expression of the five genes mentioned above. Moreover, the SeTre-1, SeG6PI, SeUAP and SeCHSB genes showed late responses to the hormone and the induction of SeTre-1, SeG6PI, SeUAP and SeCHSB genes by 20E were able to be inhibited by the protein synthesis inhibitor cycloheximide in vitro indicating these genes are 20E late-response genes.

Conclusions

We conclude that SeTre-1, SeG6PI, SeUAP and SeCHSB in the chitin biosynthesis pathway are 20E late-response genes and 20E and its specific receptors plays a key role in the regulation of chitin biosynthesis via inducing their expression.     

The POU Factor Ventral Veins Lacking/Drifter Directs the Timing of Metamorphosis through Ecdysteroid and Juvenile Hormone Signaling

Although endocrine changes are known to modulate the timing of major developmental transitions, the genetic mechanisms underlying these changes remain poorly understood. In insects, two developmental hormones, juvenile hormone (JH) and ecdysteroids, are coordinated with each other to induce developmental changes associated with metamorphosis. However, the regulation underlying the coordination of JH and ecdysteroid synthesis remains elusive. Here, we examined the function of a homolog of the vertebrate POU domain protein, Ventral veins lacking (Vvl)/Drifter, in regulating both of these hormonal pathways in the red flour beetle, Tribolium castaneum (Tenebrionidae). RNA interference-mediated silencing of vvl expression led to both precocious metamorphosis and inhibition of molting in the larva. Ectopic application of a JH analog on vvl knockdown larvae delayed the onset of metamorphosis and led to a prolonged larval stage, indicating that Vvl acts upstream of JH signaling. Accordingly, vvl knockdown also reduced the expression of a JH biosynthesis gene, JH acid methyltransferase 3 (jhamt3). In addition, ecdysone titer and the expression of the ecdysone response gene, hormone receptor 3 (HR3), were reduced in vvl knockdown larvae. The expression of the ecdysone biosynthesis gene phantom (phm) and spook (spo) were reduced in vvl knockdown larvae in the anterior and posterior halves, respectively, indicating that Vvl might influence ecdysone biosynthesis in both the prothoracic gland and additional endocrine sources. Injection of 20-hydroxyecdysone (20E) into vvl knockdown larvae could restore the expression of HR3 although molting was never restored. These findings suggest that Vvl coordinates both JH and ecdysteroid biosynthesis as well as molting behavior to influence molting and the timing of metamorphosis. Thus, in both vertebrates and insects, POU factors modulate the production of major neuroendocrine regulators during sexual maturation.     

Ecdysone differentially regulates metamorphic timing relative to 20-hydroxyecdysone by antagonizing juvenile hormone in Drosophila melanogaster

In insects, a steroid hormone, 20-hydroxyecdysone (20E), plays important roles in the regulation of developmental transitions by initiating signaling cascades via the ecdysone receptor (EcR). Although 20E has been well characterized as the molting hormone, its precursor ecdysone (E) has been considered to be a relatively inactive compound because it has little or no effect on classic EcR mediated responses. I found that feeding E to wild-type third instar larvae of Drosophila melanogaster accelerates the metamorphic timing, which results in elevation of lethality during metamorphosis and reduced body size, while 20E has only a minor effect. The addition of a juvenile hormone analog (JHA) to E impeded their precocious pupariation and thereby rescued the reduced body size. The ability of JHA impeding the effect of E was not observed in the Methoprene-tolerant (Met) and germ-cell expressed (gce) double mutant animals lacking JH signaling, indicating that antagonistic action of JH against E is transduced via a primary JH receptor, Met, or a product of its homolog, Gce. I also found that L3 larvae are susceptible to E around the time when they reach their minimum viable weight. These results indicate that E, and not just 20E, is also essential for proper regulation of developmental timing and body size. Furthermore, the precocious pupariation triggered by E is impeded by the action of JH to ensure that animals attain body size to survive metamorphosis.     

蜕皮激素与其受体EcR-USP的转录调控机制

蜕皮激素20-羟基蜕皮酮(20-hydroxyecdysone, 20E)是一种典型的类固醇激素, 主导调控昆虫的蜕皮、变态、生殖等重要生理过程。20E受体EcR-USP已被鉴定近20年, 20E与其受体复合物的转录调控机制也有了许多重要突破。已有研究表明： （1）20E受体由核受体EcR和USP形成; （2）EcR-USP异源二聚体在分子伴侣蛋白复合物的协助下获得DNA结合活性; （3）20E通过解除共阻遏因子和募集共激活因子来激活EcR USP异源二聚体并启动下游基因的转录; (4）20E-EcR-USP配体-受体复合物引发20E初级应答基因的表达, 由20E初级反应基因编码的转录因子诱导表达的20E次级应答基因级联放大20E信号, 从而调控昆虫蜕皮、变态、生殖等生理过程。     

A Critical Role of the Nuclear Receptor HR3 in Regulation of Gonadotrophic Cycles of the Mosquito Aedes aegypti

The orphan nuclear receptor HR3 is essential for developmental switches during insect development and metamorphosis regulated by 20-hydroxyecdysone (20E). Reproduction of female mosquitoes of the major vector of Dengue fever, Aedes aegypti, is cyclic because of its dependence on blood feeding. 20E is an important hormone regulating vitellogenic events in this mosquito; however, any role for HR3 in 20E-driven reproductive events has not been known. Using RNA interference (RNAi) approach, we demonstrated that Aedes HR3 plays a critical role in a timely termination of expression of the vitellogenin (Vg) gene encoding the major yolk protein precursor. It is also important for downregulation of the Target-of-Rapamycin pathway and activation of programmed autophagy in the Aedes fat body at the end of vitellogenesis. HR3 is critical in activating betaFTZ-F1, EcRB and USPA, the expressions of which are highly elevated at the end of vitellogenesis. RNAi depletion of HR3 (iHR3) prior to the first gonadotrophic cycle affects a normal progression of the second gonadotrophic cycle. Most of ovaries 24 h post second blood meal from iHR3 females in the second cycle were small with follicles that were only slightly different in length from of those of resting stage. In addition, these iHR3 females laid a significantly reduced number of eggs per mosquito as compared to those of iMal and the wild type. Our results indicate an important role of HR3 in regulation of 20E-regulated developmental switches during reproductive cycles of A. aegypti females.