Presence Of Juvenile Hormone Research Articles

Juvenile hormone membrane signaling phosphorylates USP and thus potentiates 20-hydroxyecdysone action in Drosophila

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant (Met) and germ-cell expressed (Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases. Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C (PKC) which phosphorylated ultraspiracle (USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20E through its nuclear receptor complex EcR-USP. The uspS35A mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20E signaling that delayed developmental timing. The uspS35A mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.

CREB-binding protein regulates metamorphosis and compound eye development in the yellow fever mosquito, Aedes aegypti

Juvenile hormones (JH) and ecdysone coordinately regulate metamorphosis in Aedes aegypti. We studied the function of an epigenetic regulator and multifunctional transactivator, CREB binding protein (CBP) in A. aegypti. RNAi-mediated knockdown of CBP in Ae. aegypti larvae resulted in suppression of JH primary response gene, Krüppel-homolog 1 (Kr-h1), and induction of primary ecdysone response gene, E93, resulting in multiple effects including early metamorphosis, larval-pupal intermediate formation, mortality and inhibition of compound eye development. RNA sequencing identified hundreds of genes, including JH and ecdysone response genes regulated by CBP. In the presence of JH, CBP upregulates Kr-h1 by acetylating core histones at the Kr-h1 promoter and facilitating the recruitment of JH receptor and other proteins. CBP suppresses metamorphosis regulators, EcR-A, USP-A, BR-C, and E93 through the upregulation of Kr-h1 and E75A. CBP regulates the expression of core eye specification genes including those involved in TGF-β and EGFR signaling. These studies demonstrate that CBP is an essential player in JH and 20E action and regulates metamorphosis and compound eye development in Ae. aegypti.

Recombinant yellow protein of the takeout family and albino-related takeout protein specifically bind to lutein in the desert locust

Yellow protein of the takeout family (YPT) and albino-related takeout protein (ALTO) are involved in body-color polyphenism in Schistocerca gregaria. YPT has been proposed to bind to β-carotene, whereas the physiological role of ALTO is unclear. Structurally, takeout proteins contain a long continuous tunnel to bind specific ligands. However, the specific ligands of YPT and ALTO have not been fully elucidated. Here, we isolated the full coding cDNAs of these proteins and successfully produced recombinant YPT and ALTO using an Escherichia coli expression system. Absorption spectral analyses of YPT with and without carotenoids revealed that this protein bound to lutein. In contrast, obvious binding of YPT to β-carotene and astaxanthin was not detected. Similar results were obtained for ALTO. The presence of juvenile hormone only weakly affected the protein/carotenoid interactions. These results suggested that YPT and ALTO specifically bound to lutein in a juvenile hormone-independent manner.

Cross-talk between juvenile hormone and ecdysone regulates transcription of fibroin modulator binding protein-1 in Bombyx mori.

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) are the most important hormones in silkworm and play vital roles in silkworm development, metamorphosis, and silk protein synthesis. Fibroin modulator binding protein-1 (FMBP-1) is a novel transcription factor regulating fibroin heavy chain (fib-H) transcription in Bombyx mori. The roles of JH and 20E on FMBP-1 transcription are less known. Here, we show FMBP-1 transcription is repressed by juvenile hormone analog (JHA) and activated by 20E. We identify two Krüppel homolog 1 (Kr-h1) binding sites (KBS1 and KBS2) and an E74A binding site (EBS) in the promoter of FMBP-1. We demonstrate Kr-h1 directly binds to KBS1 and KBS2 to repress FMBP-1 transcription, and 20E promotes FMBP-1 transcription through E74A. In the presence of JH and 20E, E74A abolishes the repression of Kr-h1 and activates FMBP-1 transcription through direct binding to EBS between KBS1 and KBS2 in FMBP-1 promoter. Further, JHA and 20E treatment and RNA interference confirm the effects of JH and 20E on FMBP-1 transcription in vivo, thus affecting fib-H transcription. Our results reveal the molecular mechanism of FMBP-1 transcription regulated by the cross-talk between JH and 20E in Bombyx mori, and provide novel insights into FMBP-1 transcriptional regulation and silk protein synthesis.

Juvenile hormone-regulated alternative splicing of the taiman gene primes the ecdysteroid response in adult mosquitoes

Juvenile hormone (JH) regulates many aspects of insect development and reproduction. In some processes, JH plays a critical role in defining the action of the steroid hormone 20-hydroxyecdysone (20E). In Aedes aegypti mosquitoes, JH prepares newly emerged female adults to become competent to synthesize vitellogenin in response to 20E after blood ingestion. The molecular basis of this competence is still not well understood. Here, we report that JH regulates pre-mRNA splicing of the taiman gene, which encodes a key transcriptional regulator required for both JH- and 20E-controlled gene expression. JH stimulated the production of the Taiman isoforms A/B, while reducing the levels of the isoforms C/D, in the fat body after adult eclosion. The appearance of the A/B isoforms in maturing mosquitoes was accompanied by acquisition of the competence to respond to 20E. Depletion of the A/B isoforms, by inhibiting the alternative splicing or by isoform-specific RNA interference, considerably diminished the 20E-induced gene expression after a blood meal and substantially impaired oocyte development. In accordance with this observation, further studies indicated that in the presence of 20E, the Taiman A/B isoforms showed much stronger interactions with the 20E receptor complex than the Taiman C/D isoforms. In contrast, all four isoforms displayed similar capabilities of forming active JH receptor complexes with the methoprene-tolerant protein (Met). This study suggested that JH confers the competence to newly emerged female mosquitoes by regulating mRNA splicing to generate the Taiman isoforms that are essential for the vitellogenic 20E response.

Juvenile hormone signaling - a mini review.

Since it was first postulated by Wigglesworth in 1934, juvenile hormone (JH) is considered a status quo hormone in insects because it prevents metamorphosis that is initiated by the molting hormone 20-hydroxyecdysone (20E). During the last decade, significant advances have been made regarding JH signaling. First, the bHLH-PAS transcription factor Met/Gce was identified as the JH intracellular receptor. In the presence of JH, with the assistance of Hsp83, and through physical association with a bHLH-PAS transcriptional co-activator, Met/Gce enters the nucleus and binds to E-box-like motifs in promoter regions of JH primary-response genes for inducing gene expression. Second, the zinc finger transcription factor Kr-h1 was identified as the anti-metamorphic factor which transduces JH signaling. Via Kr-h1 binding sites, Kr-h1 represses expression of 20E primary-response genes (i.e. Br, E93 and E75) to prevent 20E-induced metamorphosis. Third, through the intracellular signaling, JH promotes different aspects of female reproduction. Nevertheless, this action varies greatly from species to species. Last, a hypothetical JH membrane receptor has been predicted to be either a GPCR or a tyrosine kinase receptor. In future, it will be a great challenge to understand how the JH intracellular receptor Met/Gce and the yet unidentified JH membrane receptor coordinate to regulate metamorphosis and reproduction in insects.

Juvenile hormone-independent function of Krüppel homolog 1 in early development of water flea Daphnia pulex

Elaborate regulation of insect metamorphosis is the consequence of physiological cooperation among multiple endocrine factors such as juvenile hormones (JHs) and ecdysteroids. Hormone-induced transcription factors play important roles in substantive interactions between hormonal signaling pathways. In insects, zinc finger transcription factor Krüppel homolog 1 (Kr-h1) is a key gene of the endocrine signaling pathway in which it is directly upregulated by JH receptor Methoprene-tolerant (Met) in the presence of JH and then regulates multiple downstream factors, including components of the ecdysteroid signaling pathway. Although JH also plays a role in various biological phenomena in other arthropod species, little is known about the molecular basis of the JH signaling pathway. Here we cloned Kr-h1 from a branchiopod crustacean, Daphnia pulex, (DappuKr-h1) and analyzed its expression profile and developmental function together with consideration of its relationship to the JH signaling pathway. We suggest that DappuKr-h1 lacks JH responsiveness and regulatory relationship with the JH receptor. Moreover our loss-of-function analysis revealed that maternal mRNA of DappuKr-h1 plays a critical role in early development independent from the JH signaling pathway. These findings provide insights about whether and how the JH signaling pathway influenced evolution, leading to greater diversity in phylum Arthropoda.

Nucleoporin Nup358 facilitates nuclear import of Methoprene-tolerant (Met) in an importin β- and Hsp83-dependent manner

The bHLH-PAS transcription factor, Methoprene-tolerant (Met)1, functions as a juvenile hormone (JH) receptor and transduces JH signals by directly binding to E-box like motifs in the regulatory regions of JH response genes. Nuclear localization of Met is crucial for its transcriptional activity. Our previous studies have shown that the chaperone protein Hsp83 facilitates JH-induced Met nuclear import in Drosophila melanogaster. However, the exact molecular mechanisms of Met nuclear transport are not fully elucidated. Using DNA affinity chromatography, we have previously detected binding of the nucleoporin Nup358, in the presence of JH, to the JH response region (JHRR) sequences isolated from the Krüppel-homolog 1 (Kr-h1) promoter. Here, we have demonstrated that Nup358 regulates JH-Hsp83-induced Met nuclear localization. RNAi-mediated knockdown of Nup358 expression in Drosophila fat body perturbs Met nuclear transport during the 3 h after initiation of wandering, when the JH titer is high. The accompanying reduced expression of the transport receptor importin β in Nup358 RNAi flies could be one of the reasons accounting for Met mislocalization. Furthermore, a tetratricopeptide repeat (TPR) domain at the N-terminal end of Nup358 interacts with Hsp83 and is indispensable for Met nuclear localization. Overexpression of the TPR domain in Drosophila fat body prevents Met nuclear localization resulting in a decrease in JHRR-driven reporter activity and Kr-h1 expression. These data show that Nup358 facilitates JH-induced Met nuclear transport in a manner dependent on importin β and Hsp83.

Functional characterization of two paralogous JH receptors, methoprene-tolerant 1 and 2, in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Methoprene-tolerant (Met) is a bHLH-PAS transcription factor that functions as a core component of juvenile hormone (JH) receptors in insects. The genome of the silkworm, Bombyx mori (L.), encodes two Met paralogs, BmMet1 and BmMet2. We have previously proposed that BmMet2 forms a functional complex with steroid receptor co-activator (BmSRC) in the presence of JH and induces Kruppel-homolog 1 (BmKr-h1). In this study, we characterized BmMet2 domains and examined the role of BmMet1. A series of reporter and two-hybrid assays on BmMet2 revealed that the bHLH domain played a critical role in the binding to the JH response element of BmKr-h1 (kJHRE), the PAS-A in the transcriptional activation, and the PAS-B in the JH recognition. Furthermore, we confirmed that the BmMet2/BmSRC complex recognizes the core kJHRE (GGCCTCCACGTG). BmMet1 was found to interact with BmSRC and weakly activated the kJHRE reporter in a JH-dependent manner. The BmMet1 transcript level was much higher than the BmMet2 transcript level in the epidermis during the larval and pupal stages. By contrast, the BmMet2 transcript was hardly detected during the larval stage, but was abundant in the adult stage. These results suggest that BmMet1 is involved in the JH-dependent induction of BmKr-h1 in larvae, whereas BmMet2 is involved in adults.

Juvenile hormone-receptor complex acts on mcm4 and mcm7 to promote polyploidy and vitellogenesis in the migratory locust.

Juvenile hormone (JH), a sesquiterpenoid produced by the corpora allata, coordinates insect growth, metamorphosis, and reproduction. While JH action for the repression of larval metamorphosis has been well studied, the molecular basis of JH in promoting adult reproduction has not been fully elucidated. Methoprene-tolerant (Met), the JH receptor, has been recently shown to mediate JH action during metamorphosis as well as in vitellogenesis, but again, the precise mechanism underlying the latter has been lacking. We have now demonstrated using Met RNAi to phenocopy a JH-deprived condition in migratory locusts, that JH stimulates DNA replication and increases ploidy in preparation for vitellogenesis. Mcm4 and Mcm7, two genes in the DNA replication pathway were expressed in the presence of JH and Met. Depletion of Mcm4 or Mcm7 inhibited de novo DNA synthesis and polyploidization, and resulted in the substantial reduction of vitellogenin mRNA levels as well as severely impaired oocyte maturation and ovarian growth. By using luciferase reporter and electrophoretic mobility shift assays, we have shown that Met directly regulates the transcription of Mcm4 and Mcm7 by binding to upstream consensus sequences with E-box or E-box-like motifs. Our work suggests that the JH-receptor complex acts on Mcm4 and Mcm7 to regulate DNA replication and polyploidy for vitellogenesis and oocyte maturation.

Heat Shock Protein 83 (Hsp83) Facilitates Methoprene-tolerant (Met) Nuclear Import to Modulate Juvenile Hormone Signaling

Juvenile hormone (JH) receptors, methoprene-tolerant (Met) and Germ-cell expressed (Gce), transduce JH signals to induce Kr-h1 expression in Drosophila. Dual luciferase assay identified a 120-bp JH response region (JHRR) in the Kr-h1α promoter. Both in vitro and in vivo experiments revealed that Met and Gce transduce JH signals to induce Kr-h1 expression through the JHRR. DNA affinity purification identified chaperone protein Hsp83 as one of the proteins bound to the JHRR in the presence of JH. Interestingly, Hsp83 physically interacts with PAS-B and basic helix-loop-helix domains of Met, and JH induces Met-Hsp83 interaction. As determined by immunohistochemistry, Met is mainly distributed in the cytoplasm of fat body cells of the larval when the JH titer is low and JH induces Met nuclear import. Hsp83 was accumulated in the cytoplasm area adjunct to the nucleus in the presence of JH and Met/Gce. Loss-of-function of Hsp83 attenuated JH binding and JH-induced nuclear import of Met, resulting in a decrease in the JHRR-driven reporter activity leading to reduction of Kr-h1 expression. These data show that Hsp83 facilitates the JH-induced nuclear import of Met that induces Kr-h1 expression through the JHRR.

Sequences that direct subcellular traffic of the Drosophila methoprene-tolerant protein (MET) are located predominantly in the PAS domains

Methoprene-tolerant protein (MET) is a key mediator of antimetamorphic signaling in insects. MET belongs to the family of bHLH-PAS transcription factors which regulate gene expression and determine essential physiological and developmental processes. The ability of many bHLH-PAS proteins to carry out their functions is related to the patterns of intracellular trafficking, which are determined by specific sequences and indicate that a nuclear localization signal (NLS) or a nuclear export signal (NES) is present and active. Therefore, the identification of NLS and NES signals is fundamental in order to understand the intracellular signaling role of MET. Nevertheless, data on the intracellular trafficking of MET are inconsistent, and until now there hasn’t been any data on potential NLS and NES sequences. To analyze the trafficking of MET we designed a number of expression vectors encoding full-length MET, as well as various derivatives, that were fused to yellow fluorescent protein (YFP). Confocal microscopy analysis of the subcellular distribution of YFP–MET indicated that while this protein was localized mainly in the nucleus, it was also observed in the cytoplasm. This suggested the presence of both an NLS and NES in MET. Our work has shown that each of the two PAS domains of MET (PAS-A and PAS-B, respectively) contain one NLS and one NES sequence. Additional NES activity was present in the C-terminal fragment. The NLS activity located in PAS-B was dependent on the presence of juvenile hormone (JH), the potential ligand for MET. In contrast to this, JH didn’t seem to be required for the NLS in PAS-A to be active. However, on the basis of current knowledge about the function of PAS-A in other bHLH-PAS proteins, we suggest there might be other proteins that control the activity of the NLS and possibly the NES located in the PAS-A of MET. Thus, the intracellular trafficking of MET seems to be regulated by a rather complicated network of different factors.

Identification of specific interaction of juvenile hormone binding protein with isocitrate dehydrogenase.

Juvenile hormone (JH) is essential for multiple physiological processes: it controls larval development, metamorphosis and adult reproduction. In insect hemolymph more than 99 % of JH is bound to juvenile hormone binding protein (JHBP), which protects JH from degradation by nonspecific hydrolases and serves as a carrier to supply the hormone to the target tissues. In Galleria mellonella hemolymph, JHBP is found in a complex with lipid-binding high molecular weight proteins (HMWP) and this interaction is enhanced in the presence of JH. In this report, we present studies on the interaction of JHBP with low molecular weight proteins (LMWP) in the hemolymph. Using ligand blotting we found that JHBP interacts with a protein of about 44 kDa. To identify the protein that preferentially binds JHBP, a LMWP fraction was applied to a Sepharose-bound JHBP and, after washing, the column was eluted with free JHBP acting as a specific competitor or with carbonic anhydrase as a negative control. The eluted proteins were separated by SDS/PAGE and analyzed by mass spectrometry. Isocitrate dehydrogenase was identified as a component of the supramolecular complex of JHBP with hemolymph proteins.

Heterodimer of two bHLH-PAS proteins mediates juvenile hormone-induced gene expression

Juvenile hormone (JH) plays crucial roles in many aspects of insect life. The Methoprene-tolerant (Met) gene product, a member of the bHLH-PAS family of transcriptional regulators, has been demonstrated to be a key component of the JH signaling pathway. However, the molecular function of Met in JH-induced signal transduction and gene regulation remains to be fully elucidated. Here we show that a transcriptional coactivator of the ecdysteroid receptor complex, FISC, acts as a functional partner of Met in mediating JH-induced gene expression. Met and FISC appear to use their PAS domains to form a dimer only in the presence of JH or JH analogs. In newly emerged adult female mosquitoes, expression of some JH responsive genes is considerably dampened when Met or FISC is depleted by RNAi. Met and FISC are found to be associated with the promoter of the early trypsin gene (AaET) when transcription of this gene is activated by JH. A juvenile hormone response element (JHRE) has been identified in the AaET upstream regulatory region and is bound in vitro by the Met-FISC complex present in the nuclear protein extracts of previtellogenic adult female mosquitoes. In addition, the Drosophila homologs of Met and FISC can also use this mosquito JHRE to activate gene transcription in response to JH in a cell transfection assay. Together, the evidence indicates that Met and FISC form a functional complex on the JHRE in the presence of JH and directly activate transcription of JH target genes.

The hormonal pathway controlling cell death during metamorphosis in a hemimetabolous insect

Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, we have analyzed the apoptosis of the prothoracic gland of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. Here, we first show that B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, we demonstrate that the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal–adult transition. Finally, we have also shown that prothoracic gland degeneration is effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species.

Early events in adult eye development of the moth, Manduca sexta

The eye imaginal disc of Manduca sexta is created early in the final larval instar from the adult eye primordium, which is composed of fully differentiated cells of the larval head capsule epidermis. Concomitant with the down-regulation of the larval epidermal program, expression of broad, a marker of pupal commitment, is activated in the primordium. The cells then detach from the cuticle, fold inward, and begin to proliferate at high levels to produce the inverted, eye imaginal disc. These and other events that begin on the first day of the final larval instar appear to mark the initiation of metamorphosis. Little is known about the endocrine control of the initiation of metamorphosis in any insect. The hemolymph titer of juvenile hormone (JH) declines to low levels during this period and the presence of JH is sufficient to repress development in cultured eye primordia. However, maintenance of JH at high levels in vivo by treatment with long-lasting JH mimics has no apparent effect on early steps in eye imaginal disc development. We discuss our findings in the context of the endocrine control of metamorphosis. The initiation of metamorphosis in Manduca, and perhaps a wide range of insect species, appears to involve the overcoming of JH repression by an unidentified, nutrient-dependent, hormonal factor.

Interaction of bHLH-PAS proteins involved in juvenile hormone reception in Drosophila

The Methoprene- tolerant ( Met) bHLH-PAS gene is involved in juvenile hormone (JH) action in Drosophila melanogaster as a likely component of a JH receptor. We expressed Met in Drosophila S2 cells and explored for MET partners using pull-down assays. MET-MET interaction was found to occur. The germ- cell expressed ( gce) gene is another D. melanogaster bHLH-PAS gene with high homology to Met, and GCE formed heterodimers with MET. In the presence of JH or either of two JH agonists, MET-MET and MET-GCE formation was drastically reduced. Interaction between GCE and MET having N- or C-terminus truncations, bHLH or PAS-A domain deletions, or a point mutation in the PAS-B domain failed to occur. However, JH-dependent interaction occurred between GCE and MET having point mutations in bHLH or PAS-A. During development, changes in JH titer may alter partner binding by MET and result in different gene expression patterns.

Effects of juvenile hormone on 20‐hydroxyecdysone‐inducible EcR, HR3, E75 gene expression in imaginal wing cells of Plodia interpunctella lepidoptera

The IAL-PID2 cells derived from imaginal wing discs of the last larval instar of Plodia interpunctella were responsive to 20-hydroxyecdysone (20E). These imaginal cells respond to 20E by proliferative arrest followed by a morphological differentiation. These 20E-induced late responses were inhibited in presence of juvenile hormone (JH II). From these imaginal wing cells, we have cloned a cDNA sequence encoding a P. interpunctella ecdysone receptor-B1 isoform (PIEcR-B1). The amino acid sequence of PIEcR-B1 showed a high degree of identity with EcR-B1 isoforms of Bombyx mori, Manduca sexta and Choristoneura fumiferana. The pattern of PIEcR-B1mRNA induction by 20E was characterized by a biphasic response with peaks at 2 h and 18 h. The presence of the protein synthesis inhibitor anisomycin induced a slight reduction in level of PIEcR-B1 mRNA and prevented the subsequent declines observed in 20E-treated cells. Therefore, PIEcR-B1 mRNA was directly induced by 20E and its downregulation depended on protein synthesis. An exposure of imaginal wing cells to 20E in the presence of JH II caused an increased expression of Plodia E75-B and HR3 transcription factors but inhibited the second increase of PIEcR-B1 mRNA. These findings showed that in vitro JH II was able to prevent the 20E-induced differentiation of imaginal wing cells. This effect could result from a JH II action on the 20E-induced genetic cascade through a modulation of EcR-B1, E75-B and HR3 expression.

Hormonal regulation and functional role of Drosophila E75A orphan nuclear receptor in the juvenile hormone signaling pathway

Ecdysone and juvenile hormone (JH) are important regulators of insect growth and development. While ecdysone initiates a transition from one developmental stage to another, JH determines the nature of the transition. How these two hormones interact at the molecular level is not known. Here we report the JH inducibility of the E75A nuclear receptor encoded by the E75 early ecdysone-inducible gene. In Drosophila S2 cells, E75A transcription is specifically activated by JH at concentrations well within the physiological range found in larvae and adults. The induction is rapid and does not require a concurrent protein synthesis, and thus represents a primary hormone response. Consistent with JH regulation, E75A mRNA levels are reduced in ovaries of apterous 4 mutant adults defective in JH secretion. Expression is rescued by topical methoprene application. We further provide evidence that ectopic E75A is sufficient to perform several functions in the JH signaling pathway. First, it can down-regulate its own transcription. Second, E75A can potentiate the JH inducibility of a secondary response gene, JhI-21. Finally, in the presence of JH, E75A can repress ecdysone activation of early genes including Broad-Complex. Based on these data, we propose a model for the role of E75A in the ecdysone-JH regulatory interplay.

The 20-hydroxyecdysone-induced cellular arrest in G2 phase is preceded by an inhibition of cyclin expression

We have studied the effect of 20-hydroxyecdysone (20E) on cellular proliferation in IAL-PID2 cell line established from imaginal wing discs of Plodia interpunctella. Flow cytometry analysis demonstrated that 20E induced an arrest of cells in G2 phase. To determine whether this arrest was due to an effect of 20E on cyclin expression, we cloned two cDNA fragments, named PcycA and PcycB, encoding, respectively, Plodia cyclins A and B. Using PcycA and PcycB probes, we have demonstrated that 20E induced a sharp decrease in the levels of cyclin A and B expression. Studies of induction pattern of Plodia HR3 transcription factor by 20E revealed that its induction preceded the decrease of cyclin transcripts. An exposure of cells to 20E in the presence of juvenile hormone (JH) led to a change in the kinetic of PHR3 induction and prevented both the decline of cyclin A and B expression and the G2 arrest. This effect of JH provides an additional argument for the existence of a correlation between cyclin transcripts level and G2 arrest. For the first time in insects, these findings bring evidence that ecdysteroids regulate cellular proliferation by acting on cell cycle regulators as cyclins.