Synthetic Ecdysone Research Articles

Precocious Metamorphosis of Silkworm Larvae Infected by BmNPV in the Latter Half of the Fifth Instar.

The mulberry silkworm (Bombyx mori) is a model organism, and BmNPV is a typical baculovirus. Together, these organisms form a useful model to investigate host–baculovirus interactions. Prothoracic glands (PGs) are also model organs, used to investigate the regulatory effect of synthetic ecdysone on insect growth and development. In this study, day-4 fifth instar silkworm larvae were infected with BmNPV. Wandering silkworms appeared in the infected groups 12 h earlier than in the control groups, and the ecdysone titer in infected larvae was significantly higher than that of the control larvae. We then used RNA sequencing (RNA-seq) to analyze silkworm PGs 48 h after BmNPV infection. We identified 15 differentially expressed genes (DEGs) that were classified as mainly being involved in metabolic processes and pathways. All 15 DEGs were expressed in the PGs, of which Novel01674, BmJing, and BmAryl were specifically expressed in the PGs. The transcripts of BmNGDN, BmTrypsin-1, BmACSS3, and BmJing were significantly increased, and BmPyd3, BmTitin, BmIGc2, Novel01674, and BmAryl were significantly decreased from 24 to 72 h in the PGs after BmNPV infection. The changes in the transcription of these nine genes were generally consistent with the transcriptome data. The upregulation of BmTrypsin-1 and BmACSS3 indicate that these DEGs may be involved in the maturation process in the latter half of the fifth instar of silkworm larvae. These findings further our understanding of silkworm larval development, the interaction between BmNPV infection and the host developmental response, and host–baculovirus interactions in general.

Molecular Evidence for a Functional Ecdysone Signaling System in Brugia malayi

BackgroundFilarial nematodes, including Brugia malayi, the causative agent of lymphatic filariasis, undergo molting in both arthropod and mammalian hosts to complete their life cycles. An understanding of how these parasites cross developmental checkpoints may reveal potential targets for intervention. Pharmacological evidence suggests that ecdysteroids play a role in parasitic nematode molting and fertility although their specific function remains unknown. In insects, ecdysone triggers molting through the activation of the ecdysone receptor: a heterodimer of EcR (ecdysone receptor) and USP (Ultraspiracle).Methods and FindingsWe report the cloning and characterization of a B. malayi EcR homologue (Bma-EcR). Bma-EcR dimerizes with insect and nematode USP/RXRs and binds to DNA encoding a canonical ecdysone response element (EcRE). In support of the existence of an active ecdysone receptor in Brugia we also cloned a Brugia rxr (retinoid X receptor) homolog (Bma-RXR) and demonstrate that Bma-EcR and Bma-RXR interact to form an active heterodimer using a mammalian two-hybrid activation assay. The Bma-EcR ligand-binding domain (LBD) exhibits ligand-dependent transactivation via a GAL4 fusion protein combined with a chimeric RXR in mammalian cells treated with Ponasterone-A or a synthetic ecdysone agonist. Furthermore, we demonstrate specific up-regulation of reporter gene activity in transgenic B. malayi embryos transfected with a luciferase construct controlled by an EcRE engineered in a B. malayi promoter, in the presence of 20-hydroxy-ecdysone.ConclusionsOur study identifies and characterizes the two components (Bma-EcR and Bma-RXR) necessary for constituting a functional ecdysteroid receptor in B. malayi. Importantly, the ligand binding domain of BmaEcR is shown to be capable of responding to ecdysteroid ligands, and conversely, ecdysteroids can activate transcription of genes downstream of an EcRE in live B. malayi embryos. These results together confirm that an ecdysone signaling system operates in B. malayi and strongly suggest that Bma-EcR plays a central role in it. Furthermore, our study proposes that existing compounds targeting the insect ecdysone signaling pathway should be considered as potential pharmacological agents against filarial parasites.

Inducible Expression of a Constitutively Active Mutant of Mitogen-activated Protein Kinase Kinase 7 Specifically Activates c-JUN NH2-terminal Protein Kinase, Alters Expression of at Least Nine Genes, and Inhibits Cell Proliferation

MKK7 is a recently discovered mitogen-activated protein kinase (MAPK) kinase that is unique in that it specifically activates only the c-JUN NH(2)-terminal protein kinase (JNK) family of enzymes. Very little is known about the biological role of MKK7. We generated inducible cell lines from the human embryonal kidney carcinoma cell line, HEK293, by stable transfection with a constitutively active mutant of MKK7, MKK7(3E), fused to green fluorescent protein (GFP), under the control of an ecdysone-inducible promoter. Treatment of cells with the synthetic ecdysone analog ponasterone A induced expression of GFP-MKK7(3E) and resulted in sustained activation of endogenous JNK, but neither of the other endogenous MAPKs, ERK or p38. Red and green fluorescing cDNA copies of mRNA extracted from cells obtained before and after induction of GFP-MKK7(3E) were hybridized to microarrays containing more than 6,000 cDNAs in eight independent experiments. By selection criteria, 23 genes were differentially regulated after 24 h of induction of GFP-MKK7(3E) and 16 after 48 h. The expression of 9 genes was consistently changed after both 24 and 48 h of induction. These changes included down-regulation of three genes, c-myc, angiopoietin-2, and glucose-regulated protein 58, and up-regulation of 6 genes, tissue factor pathway inhibitor-2, GRP78, autotaxin, PPP1R7, the DKFZ cDNA p434D0818, and 1 unknown gene. Consistent with previously described roles of several of the altered genes, MKK7(3E) inhibited cell proliferation. These data implicate active MKK7 in the negative regulation of cell proliferation and provide evidence for a new role for this kinase in the regulation of a distinct, hitherto unrecognized set of genes.

Modulation of integrin signal transduction by ILKAP, a protein phosphatase 2C associating with the integrin-linked kinase, ILK1.

ILKAP, a protein serine/threonine (S/T) phosphatase of the PP2C family, was isolated in a yeast two-hybrid screen baited with integrin-linked kinase, ILK1. Association of ILK1 and ILKAP was independent of the catalytic activity of either partner, as assayed in co-precipitation and two-hybrid experiments. Condi tional expression of ILKAP in HEK 293 cells resulted in selective inhibition of ECM- and growth factor-stimulated ILK1 activity, but did not inhibit Raf-1 kinase activity. A catalytic mutant of ILKAP, H154D, did not inhibit ILK1 kinase activity. Two cellular targets of ILK1, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase B (PKB)/AKT, were differentially affected by ILKAP-mediated inhibition of ILK1. Catalytically active, but not mutant ILKAP, strongly inhibited insulin-like growth factor-1-stimulated GSK3beta phosphorylation on Ser9, but did not affect phosphorylation of PKB on Ser473, suggesting that ILKAP selectively affects ILK-mediated GSK3beta signalling. Consistent with this, active, but not H154D mutant or the related PP2Calpha, selectively inhibited transactivation of a Tcf/Lef reporter gene, TOPFlash, in 293 cells. We propose that ILKAP regulates ILK1 activity, targeting ILK1 signalling of Wnt pathway components via modulation of GSK3beta phosphorylation.

Transcriptional activation of the Drosophila ecdysone receptor by insect and plant ecdysteroids

A number of insect ecdysteroids, plant ecdysteroids and juvenoids were assayed for their ability to activate Drosophila nuclear receptors in transfected tissue culture cells. Discrete modifications to 20-hydroxyecdysone, the apparent natural ligand for the ecdysone receptor (EcR), conferred dramatic changes on the transcriptional activity of this receptor, suggesting that other biologically relevant EcR ligands may exist. Conversely, none of the compounds tested had a significant effect on the activity of three Drosophila orphan nuclear receptors: DHR38, DHR78 or DHR96. Taken together, these results demonstrate the selectivity of EcR for a series of natural and synthetic ecdysone agonists and suggest that as yet untested compounds may be responsible for activating DHR38, DHR78 and DHR96.

DNA Binding and Transactivation Characteristics of the Mosquito Ecdysone Receptor-Ultraspiracle Complex

The steroid hormone 20-hydroxyecdysone is a key regulatory factor, controlling blood-meal triggered egg maturation in mosquitoes. To elucidate the ecdysone hierarchy governing this event, we cloned and characterized the ecdysone receptor (AaEcR) and the nuclear receptor Ultraspiracle (AaUSP), a retinoid X receptor homologue, from the mosquito, Aedes aegypti, which form a functional complex capable of ligand and DNA binding. Here we analyzed the DNA-binding properties of the AaEcR.AaUSP heterodimer with respect to the effects of nucleotide sequence, orientation, and spacing between half-sites in natural Drosophila and synthetic ecdysone response element (EcREs). By using an electrophoretic gel mobility shift assay, we showed that AaEcR.AaUSP exhibits a broad binding specificity, forming complexes with inverted (IR) and direct (DR) repeats of the nuclear receptor response element half-site consensus sequence AGGTCA separated by spacers of variable length. A single nucleotide spacer was optimal for both imperfect (IRhsp-1) and perfect (IRper-1) inverted repeats; adding or removing 1 base pair in an IRhsp-1 spacer practically abolished binding. However, changing the half-site to the consensus sequence AGGTCA (IRper-1) increased binding of AaEcR.AaUSP 10-fold over IRhsp-1 and, at the same time, reduced the stringency of the spacer length requirement, with IRper-0 to IRper-5 showing detectable binding. Spacer length was less important in DRs of AGGTCA (DR-0 to DR-5); although 4 bp was optimal, DR-3 and DR-5 bound AaEcR.AaUSP almost as efficiently as DR-4. Furthermore, AaEcR. AaUSP also bound DRs separated by 11-13 nucleotide spacers. Competition experiments and direct estimation of binding affinity (Kd) indicated that, given identical consensus half-sites and an optimal spacer, the AaEcR.AaUSP heterodimer bound an IR with higher affinity than a DR. Co-transfection assays utilizing CV-1 cells demonstrated that the mosquito EcR.USP heterodimer is capable of transactivating reporter constructs containing either IR-1 or DR-4. The levels of transactivation are correlated with the respective binding affinities of the response elements (IRper-1 > DR-4 > IRhsp-1). Taken together, these analyses predict broad variability in the EcREs of mosquito ecdysone-responsive genes.

High level transactivation by the ecdysone receptor complex at the core recognition motif.

Ecdysteroid signaling in insects is mediated by the ecdysone receptor complex that is composed of a heterodimer of the ecdysone receptor and Ultraspiracle. The DNA binding specificity plays a critical role of defining the repertoire of target genes that respond to the hormone. We report here the determination of the preferred core recognition motif by a binding site selection procedure. The consensus sequence consists of a perfect palindrome of the heptameric half-site sequence GAGGTCA that is separated by a single A/T base pair. No binding polarity of the ecdysone receptor/Ultraspiracle heterodimer to the core recognition motif was observed. This core motif mediated the highest level of ligand-induced transactivation when compared to a series of synthetic ecdysone response elements and to the natural element of the Drosophila hsp27 gene. This is the first report of a palindromic sequence identified as the highest affinity DNA binding site for a heterodimeric nuclear hormone receptor complex. We further present evidence that the ligand of the ecdysone receptor preferentially drives Ultraspiracle from a homodimer into a heterodimer. This mechanism might contribute additionally to a tight control of target gene expression.

Identification of ecdysone 22-long-chain fatty acyl esters in newly laid eggs of the cattle tick Boophilus microplus.

The five major apolar ecdysone esters present in newly laid eggs of the cattle tick Boophilus microplus have been purified by h.p.l.c. The quantities of the apolar esters present in the eggs were increased by administration of ecdysone to the mature females. G.c.-m.s. analysis, as their methyl esters, of the fatty acids released from the apolar ecdysone derivatives by alkali, coupled with positive-ion fast-atom-bombardment m.s. of the intact ecdysone esters, showed that the compounds consisted of a series of fatty acyl esters of ecdysone. The position of esterification of the ecdysone was established by p.m.r. spectroscopy. The combined data show that the novel apolar derivatives of ecdysone consist of the 22-palmitate, -palmitoleate, -stearate, -oleate, and -linoleate esters respectively. Confirmation was obtained by comparison with synthetic ecdysone 22-palmitate. The significance of the ecdysone fatty acyl esters as a possible source of free hormone during embryogenesis is discussed.

Effect of administration of synthetic ecdysone on the moulting ofPalamnaeus bengalensis

Synthetic analogue of ecdysone, 3β, 14α-dihydroxy-5β-cholest-7-en-6-one did not bring the scorpionPalamnaeus bengalensis to moult. It also failed completely in stimulating the scorption to produce any change related to moulting.

Effects of ecdysones and some synthetic analogs on horseshoe crab larvae

The two insect molting hormones, α- and β-ecdysone, and two synthetic ecdysone analogs are highly effective in stimulating molting in horseshoe crab ( Limulus) larvae. Effectiveness of the ecdysones is dependent on time during the intermolt cycle. Large doses were necessary to induce molting early in the cycle and no larvae completed ecdysis, but late in the cycle only 0.02 – 0.06 μg/g of the natural ecdysones were necessary for a 50% response, and over 80% of the injected larvae completed ecdysis. Responses of Limulus larvae to the ecdysones and some synthetic analogs suggest some similarities to the chemical control of molting in an insect (house fly larvae).

Synthesis and biological activity of 5β-hydroxy analogs of α-ecdysone

Two 5β-hydroxy analogs of α-ecdysone — 2β,3β,5β,14α-tetrahydroxy-5β-cholest-7-en-6-one and 3β,5β,14α-trihydroxy-5β-cholest-7-en-6-one were synthesized and their mass spectra obtained. The biological activity of these two compounds was compared with that of the most active synthetic ecdysone analog previously tested in insects, and the presence of the 5β-hydroxyl group generally enhanced the inhibitive activity.

Ecdysone Analog: Conversion to Alpha Ecdysone and 20-Hydroxyecdysone by an Insect

The tritium-labeled synthetic ecdysone analog Delta(7)-5beta-cholestene-2beta,-3beta,14alpha-triol-6-one terminated diapause when injected into diapausing tobacco hornworm pupae and was converted into tritium-labeled alpha-ecdysone and tritium-labeled 20-hydroxyecdysone. About half of the crystalline alpha-ecdysone and 20-hydroxyecdysone isolated from the tobacco hornworms 7(1/2) to 8(1/2) days after injection was derived from endogenous steroid precursors and half from the transformation of the synthetic ecdysone analog.

Ecdysones and Analogs: Effects on Development and Reproduction of Insects

Ingestion of certain synthetic ecdysone analogs inhibited larval growth and development in several species of insects, whereas 20-hydroxyecdysone was inactive or considerably less active. Natural 20-hydroxyecdysone and ponasterone A, and a synthetic ecdysone analog inhibited ovarian maturation and egg production in the adult housefly. These effects appeared to be related to hormonal activity.

Active transport by the cecropia midgut. V. Loss of potassium transport during larval-pupal transformation.

ABSTRACT The electrical potential across the isolated midgut of five developmental stages of the Cecropia silkworm was studied by changing the concentration of single cations in solutions bathing each side of the midgut. The stages included feeding fourth-instar insects, insects moulting from the fourth to the fifth instar, feeding fifth-instar insects, insects which had evacuated their midguts, and insects spinning cocoons. Average values of the initial maximal potential exhibited by the midgut in solutions containing K, Mg, and Ca but no Na, for the stages mentioned above, were 68, 83, 90, 124, and 2 mV., respectively. In all of the developmental stages studied except the ‘spinning larva’, reducing the potassium concentration from 32 to 2 mM/l. on the blood-side of the isolated gut lowers the potential, on the lumen-side of the gut raises the potential and on both sides gives an intermediate value. When the potential prior to a decrease in concentration of potassium on the blood-side is over 100 mV., the Nernst slope approaches 59 mV. A tenfold reduction in the concentration of magnesium or the addition of 32 mM/l. sodium to the solutions bathing the isolated gut has no systematic effect on the potential. A tenfold drop in the concentration of calcium in the solutions causes changes in the potential in the opposite direction from those predicted by the Nernst equation. The pH of the midgut contents rises from early fourth instar to late fifth instar. The hydrogen-ion concentration of the blood is about 1000 times more than that of midgut contents in fifth-instar insects. Neither synthetic ecdysone, partially purified natural ecdysone nor juvenile hormone has an effect on the potential or current of the isolated midgut over periods as long as 30 min.