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Insect Biochemistry and Molecular Biology 38 (2008) 529–539

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The 20-hydroxyecdysone-induced signalling pathway in G2/M arrestof Plodia interpunctella imaginal wing cells

David Siaussat, Franc-oise Bozzolan, Patrick Porcheron, Stephane Debernard�

UMR 1272A Physiologie de l’Insecte, Signalisation et Communication, Universite Pierre et Marie Curie, 7 quai Saint Bernard, 75005 Paris, France

Received 18 September 2007; received in revised form 10 January 2008; accepted 11 January 2008

Abstract

The mechanisms involved in the control of cellular proliferation by the steroid hormone 20-hydroxyecdysone (20E) in insects are not

known. We dissected the 20E signalling pathway responsible for G2/M arrest of imaginal cells from the IAL-PID2 cells of the Indian

meal moth Plodia interpunctella. We first used a 50–30 RACE-based strategy to clone a 4479 bp cDNA encoding a putative

P. interpunctella HR3 transcription factor named PiHR3. The deduced amino acid sequence of PiHR3 was highly similar to those of

HR3 proteins from other lepidopterans, e.g. Manduca sexta and Bombyx mori. Using double-stranded RNA-mediated interference

(dsRNAi), we then succeeded in blocking the ability of 20E to induce the expression of PiEcR-B1, PiUSP-2 and PiHR3 genes that encode

the P. interpunctella ecdysone receptor B1-isoform, Ultraspiracle-2 isoform, the insect homologue of the vertebrate retinoid X receptor,

and the HR3 transcription factor. We showed that inhibiting the 20E induction of PiEcR-B1, PiUSP-2 and PiHR3 mRNAs prevented

the decreased expression of B cyclin and consequently the G2/M arrest of IAL-PID2 cells. Using this functional approach, we revealed

the participation of EcR, USP and HR3 in a 20E signalling pathway that controls the proliferation of imaginal cells by regulating the

expression of B cyclin.

r 2008 Elsevier Ltd. All rights reserved.

Keywords: Steroid hormone receptor superfamily; G2/M arrest; RNA interference; B cyclin; Signalling pathway; Imaginal cells

1. Introduction

In higher organisms, steroid hormones control a varietyof cellular functions, including cell proliferation, expan-sion, differentiation and programmed cell death (Hong etal., 2007; Vujovic et al., 2007; Li et al., 2007; Hsieh et al.,2007; Gao et al., 2006; Kilpatrick et al., 2005; Champlinand Truman, 1998; Siaussat et al., 2004a, b, 2007). Ininsects, the steroid hormone 20-hydroxyecdysone (20E),which belongs to the ecdysteroid family, is responsible forthe radical reorganization of the body form that occurs atmetamorphosis (Riddiford and Truman, 1993). At thistime, 20E activates genetic cascades that lead to both thehistolysis of larval structures and the morphogenesis ofadult structures by the differentiation of diploid imaginalcells. Most 20E-induced biological effects have beenassociated with genomic mechanisms that involve a

e front matter r 2008 Elsevier Ltd. All rights reserved.

mb.2008.01.001

ing author. Tel.: +331 44 27 65 93; fax: +33 1 44 27 65 09.

ess: stephane.debernard@snv.jussieu.fr (S. Debernard).

hormone binding to a nuclear heterodimeric complexcomposed of the ecdysone receptor (EcR) (Koelle et al.,1991; Schubiger et al., 1998) and Ultraspiracle (USP) (Yaoet al., 1992; Swevers et al., 1996), the insect homologue ofthe vertebrate retinoid X receptor (RXR) (Mangelsdorfet al., 1992, 1995). The 20E/EcR/USP complex triggers thesequential expression of genes encoding transcriptionfactors such as HR3, E74, E75, which ultimately regulatethe activity of target genes (Henrich et al., 1999; Andresand Thummel, 1992; Thummel, 1995; Thummel et al.,1990; Koelle et al., 1992; Segraves and Hogness, 1990).Some developmental studies have shown that EcR andUSP exist as isoforms that are expressed in a tissue-specificand a stage-specific manner, thus contributing to thespatial and temporal diversity of the response to 20E(Perera et al., 1999; Talbot et al., 1993; Bender et al., 1997;Jindra et al., 1996; Schubiger et al., 1998).The imaginal discs are tucked away in the body of the

larva and contribute little or nothing to its functioning(Cohen, 1993). In lepidopteran larvae, the rate of

ARTICLE IN PRESSD. Siaussat et al. / Insect Biochemistry and Molecular Biology 38 (2008) 529–539530

proliferation of the imaginal cells is fairly constant in theyoung instars. At the end of the last larval instar, however,many of the imaginal cells are blocked in the G2/M phasein response to the rising ecdysteroid titre precedingpupation (Kurushima and Ohtaki, 1975; Meyer et al.,1980; Oberlander, 1985; Fain and Stevens, 1982; Gravesand Schubiger, 1982). This arrest coincides with thebeginning of pupal differentiation of imaginal discs intoadult organs to replace the larval counterpart. On the otherhand, this 20E pulse is correlated with a high level ofinduction of EcR, USP and HR3 mRNAs (Fujiwara et al.,1995; Jindra et al., 1996). These data reveal a 20Einteraction with the cell-cycle and suggest that a quantita-tive increase of EcR, USP and HR3 transcripts is somehowessential in inducing G2/M arrest. Nevertheless, themolecular mechanisms involved in the control of cell-cycleby 20E are not known.

The establishment of various insect cell lines that haveretained sensitivity to 20E provided new potential forspecifying the mode of action of ecdysteroids in theregulation of cellular processes. The Indian meal moth(Plodia interpunctella) cell line IAL-PID2 has beenestablished from pupally committed imaginal wing discs(Lynn and Oberlander, 1983). The IAL-PID2 cells respondto treatment with 20E by the arrest of cell growth and long-term spectacular morphological transformations (Coudercet al., 1987; Dinan et al., 1990; Siaussat et al., 2004a, b).Fluorescent flow cytometry analysis of the P. interpunctella

IAL-PID2 cell-cycle have shown that 20E-induced prolif-erative arrest results from a blockage of cells in the G2/Mphase (Siaussat et al., 2004a; Mottier et al., 2004; Stevens etal., 1980). The cell-cycle is regulated by the action of afamily of serine/threonine kinases known as cdks. Differentcdks function at different times of the cell-cycle and arepositively regulated through binding to subunits calledcyclins, a process that is essential for cdk activity (Buttittaet al., 2007; Meyer et al., 2000; Boylan and Gruppuso,2005; Saab et al., 2006). It has been reported that the 20E-induced G2/M arrest in IAL-PID2 cells was preceded by adecrease in the level of expression of B cyclin, which isinvolved in the control of the G2/M transition of cell-cycle(Mottier et al., 2004). Only descriptive information existsfor the regulation of the cyclin gene by ecdysteroids incorrelation with G2/M arrest, and there are no functionaldata on the 20E signalling transduction pathway respon-sible for these genetic and cellular responses. Recentexperiments have highlighted the efficacy of double-stranded RNA-mediated interference (dsRNAi) on thesteroid hormone-inducible gene expression in IAL-PID2cells (Siaussat et al., 2007). Therefore, this 20E responsivecell line seemed to be an ideal system to undertake theidentification of the signalling pathway responsible forG2/M arrest of imaginal cells by dsRNAi. We first isolateda cDNA fragment encoding a putative P. interpunctella

HR3 transcription factor (PiHR3). We specifically blockedthe 20E inducibility of P. interpunctella ecdysone re-ceptor B1-isoform (PiEcR-B1), Ultraspiracle-2 isoform

(PiUSP-2), and HR3, and reported the consequences ofthese knockdowns on the level of expression of B cyclinand the induction level of G2/M arrest. This functionalanalysis provided evidence of the involvement of EcR, USPand HR3 in a common ecdysteroid signalling pathway thatcontrols the proliferation of imaginal cells by regulating thesynthesis of B cyclin.

2. Materials and methods

2.1. Cell culture

IAL-PID2 cell line was established from imaginal wingdiscs of last instar larval of P. interpunctella Hubner, theIndian meal moth (Lynn and Oberlander, 1983). The cellline kept its sensitivity to 20E. Cells grow as a looselyattached monolayer. We maintained them at 26 1C in75 cm2 tissue culture flasks with 12ml of antibiotic-freeGrace’s medium (Gibco BRL, Life Technologies, Inc.)supplemented with 10% heat-inactivated foetal bovineserum (FBS) (Boerhinger Mannheim, France) and 1%bovine serum albumin (BSA) (Calbiochem, France). Cellswere subcultured weekly to a near confluent monolayer.Cells were rinsed off the bottom of the flask in a gentlestream of culture medium and resuspended. Cell densitywas estimated by counting the cells in an aliquot of the sus-pension in a Mallassez hemocytometer under the micro-scope. All the cultures were initiated by seeding flasks with1.5� 106 cells and cultured under normal growth condi-tions for 72 h. 20E was a gift of Dr. Rene Lafont (UPMC,Paris, France). For use in culture, 20E was dissolved inethanol and diluted in appropriate volumes of sterileGrace’s medium supplemented with FBS and BSA. Then,the solution was added directly to cell cultures by usingglass capillary pipets. Final ethanol concentration in alltreatments and control cultures was maintained under0.1% in order to prevent any toxic effect of the solvent.

2.2. Preparation of double-stranded RNA (dsRNA) and

dsRNA interference experiments

The MEGAscriptTM RNAi kit (Ambion, www.Am-bion.com) was used to generate dsRNAs corresponding toRpL8, P. interpunctella B cyclin (PiCycB), the specific A/Bregion of PiEcR-B1, PiUSP-2 and specific C region ofPiHR3. T7 promoter sites were added to the PCR primersRPD 50-forward primer (50-GCG ATC TTA GGC GCATGC TTG-30), RPR 50-reverse primer (50-ATT GGC CCGACG TGG CGC ATC-30) for RpL8; CYD 50-forwardprimer (50-CGG TGG ATT CCA TGC CCA CGT-30),50-forward primer CYR (50-ATT CGC GGC TGG TTACCC CTG-30) for CycB; CED 50-forward primer (50-CGCTGG TCC AAC AAC GGA GGG-30), CER 50-reverseprimer (50-TGC CGG TGA CAA CTC CTC ACG-30) forEcR-B1; USD3 50-forward primer (50-ATG GAG CCCTCG AGG GAA TCA-30), USR3 50-reverse primer(50-AGA CGT GGC GTC GTC CCG GAG-30) for

ARTICLE IN PRESSD. Siaussat et al. / Insect Biochemistry and Molecular Biology 38 (2008) 529–539 531

USP-2 and A11 50-forward primer (50-CCG TGC AAAGTT TGC GGC G-30), A12 50-reverse primer (50-GCTCAT GCC GAG TTT GAG GC-30) for HR3. Thegenerated primers were T7 RPD (50-TAA TAC GACTCA CTA TAG GGA GCG ATC TTA GGC GCA TGCTTG), T7 RPR (50-ATT ATG CTG AGT GAT ATC CCTATT GGC CCG ACG TGG CGC ATC) for RpL8; T7CYD (50-TAA TAC GAC TCA CTA TAG GGA CGGTGG ATT CCA TGC CCA CGT), T7 CYR (50-ATT ATGCTG AGT GAT ATC CCT ATT CGC GGC TGG TTACCC CTG-30) for CycB; T7 CED (50-TAA TAC GACTCA CTA TAG GGA CGC TGG TCC AAC AAC GGAGGG-30), T7 CER (50-ATT ATG CTG AGT GAT ATCCCT TGC CGG TGA CAA CTC CTC ACG-30) forEcR-B1; T7 USD3 (50-TAA TAC GAC TCA CTA TAGGGA ATG GAG CCC TCG AGG GAA TCA-30), T7URD3 (50-ATT ATG CTG AGT GAT ATC CCT AGACGT GGC GTC GTC CCG GAG-30) for USP-2 and T7A11 (50-TAA TAC GAC TCA CTA TAG GGA CCGTGC AAA GTT TGC GGC G-30) and T7 A12 (50-ATTATG CTG AGT GAT ATC CCT GCT CAT GCC GAGTTT GAG GC-30) for HR3. Separate PCR reactions wereconducted to generate complementary templates with asingle T7 promoter site (T7 RPR+RPD, RPR+T7 RPD,T7 CYD+CYR, CYD+T7 CYR, T7 CED+CER,CED+T7 CER; T7 USD3+USR3; USD3+T7 USR3and T7 A11+A12, A11+T7 A12). T7 RNA polymerasewas used to transcribe single-stranded RNA from eachDNA template over 4 h at 37 1C. The dsRNAs (RpL8dsRNA, CycB dsRNA, EcR-B1 dsRNA, USP-2 dsRNA,HR3 dsRNA) were produced by mixing solutions contain-ing equivalent amounts of complementary ssRNA at 90 1Cfor 30min followed by slow cooling to room temperature.DNA and single-stranded RNA were removed from thesolution by digestion with DNase I and Rnase A at 37 1Cfor 1 h. The dsRNAs were precipitated with ethanol andresuspended in nuclease-free water. The dsRNA concen-tration was determined by spectrophotometry at 260 nm.Six micrograms of dsRNA were analysed by 1% agaroseelectrophoresis to ensure that the majority of the dsRNAexisted as a single band of expected size. The dsRNAs werestored at �20 1C for several months. For RNAi experi-ments, the preparation of dsRNA-Lipofectamine 2000complexes and the transfection into IAL-PID2 cells aredescribed by Siaussat et al. (2007).

2.3. Determination of cell-cycle stage by FACS

Cell DNA content was determined by staining cells withpropidium iodide and measuring fluorescence (FACS,Institut Jacques Monod, Paris, France). The IAL-PID2cells were resuspended and fixed in cool 30% PBS (10mMNa2HPO4, 138mM NaCl, 2.7mM KCl, pH 7.4). Subse-quently, the fixed cells were incubated in a solutioncontaining 1mg/ml RNase and 400 mg/ml propidium iodidefor 30min at 37 1C. For each cell population, 10,000 cellswere analysed by FACS and the percentage of cells in a

specific phase of the cell-cycle was determined with thepropidium iodide DNA staining technique. Cells wereclassified in G0/G1, G2/M and S stages depending on theintensity of the fluorescence peaks (Crissman et al., 1975).

2.4. Isolation of RNA and cDNA synthesis

Total RNAs from cells were extracted with TRIzolreagent (Gibco, BRL) and quantified by spectrophoto-metry at 260 nm. The quality of RNA was checkedby electrophoresis on formaldehyde-agarose gel (1%).For the 50 and 30 RACE, cDNA was synthetized from1 mg of total RNAs at 42 1C for 1.5 h using the SMARTRACE cDNA Amplification kit (Clontech) with 200U ofSuperscript II (GibcoBRL), 50 or 30CDS-primer andSMART II oligonucleotide, according to the instructionsin the kit.

2.5. Rapid amplification of cDNA 50/30 terminal ends

(50/30 RACE)

A 204 bp cDNA fragment encoding the zinc fingerdomain of P. interpunctella PiHR3 has been already clonedand characterized in our laboratory (Debernard et al.,2001). The 50 and 30 regions of the corresponding cDNAwere obtained by 50 and 30 RACE (SMART RACE cDNAamplification kit) following the manufacturer’s instruc-tions. For 50 RACE, we used 2 ml of 50 RACE-ready cDNAwith a specific reverse primer 50 Race PHR (50-CTG ACATCT GTT GCG GTT CAC-30) and Universal Primer Mix(UPM, Clontech) as the forward anchor primer. The 30-RACE amplification was carried out with UPM as thereverse primer and a specific forward primer 30 Race PHD(50-GGG GTG ATC ACG TGC GAA GGA-30). Touch-down PCR was performed using hot start as follows: after1min at 94 1C, five cycles of 30 s at 94 1C and 5min at72 1C, then five cycles of 30 s at 94 1C, 30 s at 70 1C and3min at 72 1C, then 25 cycles of 30 s at 94 1C, 30 s at 68 1Cand 5min at 72 1C, then 7min at 72 1C. The 30 and 50 Racefragments were purified and cloned as described earlier.The analysis of the overlapping sequences of these twoclones with the cDNA encoding the zinc finger domain ofHR3 P. interpunctella allowed us to obtain a 4479-bpcDNA fragment named PiHR3.

2.6. Generation of DIG-labelled probe

PiCycB, PiEcR-B1, PiUSP-2 and PiHR3 cDNAs werelabelled with Digoxigenin (DIG) by PCR using the PCRDIG probe synthesis kit (Roche, Meylan, France) withCYD 50-forward primer (50-CGG TGG ATT CCA TGCCCA CGT-30), 50-reverse primer CYR (50-ATT CGC GGCTGG TTA CCC CTG-30) for CycB; CED 50-forwardprimer (50-CGC TGG TCC AAC AAC GGA GGG-30),CER 50-reverse primer (50-TGC CGG TGA CAA CTCCTC ACG-30) for EcR; USD3 50-forward primer (50-ATGGAG CCC TCG AGG GAA TCA-30), USR3 50-reverse

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Table 1

Comparison of amino acid sequences of C and E/F domains between

PiHR3 and homologues isolated from Manduca sexta HR3 (MsHR3),

Bombyx mori HR3 (BmHR3), Aedes aegypti HR3 (AaHR3) and

Drosophila melanogaster HR3 (DmHR3)

C domain E/F domain

D. Siaussat et al. / Insect Biochemistry and Molecular Biology 38 (2008) 529–539532

primer (50-AGA CGT GGC GTC GTC CCG GAG-30) forUSP and A11 50-forward primer (50-CCG TGC AAA GTTTGC GGC G-30), A12 50-reverse primer (50-GCT CATGCC GAG TTT GAG GC-30) for HR3. The DIG-labelledprobe was used at a concentration of 25 ng/ml in thehybridization solution.

Identity (%) Length (amino acids)

PiHR3 100 66 100 249

BmHR3 100 64 90 249

AaHR3 97 62 68 248

MsHR3 100 64 90 249

DmHR3 97 62 67 248

The length of C and E/F domains of the HR3 nuclear receptors (no. of

amino acids) are indicated and the identity vs. PiHR3 is expressed as a

percentage of the PiHR3 sequence.

2.7. Northern blotting

Northern blot hybridization analysis was performedaccording to the manufacturer’s instructions. RNA sam-ples (15 mg) were denatured with formamide (50%) andformaldehyde (2.2M), separated on 1% denaturatingagarose gel and transferred to a Boerhinger Mannheimpositively charged nylon membrane. Blotted RNA washybridized overnight at 55 1C with the PiEcR-B1 or PiUSP-2 or PiCycB probe and at 45 1C with the PiHR3 probe. ADIG-labelled fragment of the cDNA encoding the RpL8ribosomal protein of P. interpunctella was used as con-trol probe. An immunological signal detection by chelu-minescence was performed as described in Roche’s DIGsystem User’s Guide for filter hybridization. A molecularRNA marker ladder DIG-labelled (Roche, Meylan,France) was run in parallel on Northern blot to determinethe molecular weight of hybridizing RNAs. The relativemRNA expression levels were quantified from the North-ern blots by the molecular imager system, Model Bio-1D(Vilber Lourmat).

3. Results

3.1. Isolation and characterization of P. interpunctella HR3

mRNA

A partial 204 bp cDNA fragment encoding the DNA-binding domain of P. interpunctella HR3 has been clonedfrom IAL-PID2 cells (Debernard et al., 2001). Using a 50–30

RACE-based strategy, we succeeded in isolating the 50 and30 ends of the corresponding cDNA. By merging theoverlap of the sequences obtained, a full 4479 bp cDNAfragment was generated and named PiHR3. The nucleotidesequence and the amino acid sequence of PiHR3 have beendeposited in GenBank under the accession numberAY573570. The 548 amino acid residue region of PiHR3showed 76%, 73%, 57% and 56% identity with Manduca

sexta HR3 (MsHR3; Palli et al., 1992), Bombyx mori HR3(BmHR3; Eystathioy et al., 2001), Aedes aegypti HR3(AaHR3; Kapitskaya et al., 2000) and Drosophila melano-

gaster HR3 (DmHR3; Koelle et al., 1992), respectively(Fig. S1). In addition, a high level of amino acid identitywas observed with MsHR3, BmHR3, AeHR3 and DmHR3in both the DNA-binding (C) domain and the ligand-bind-ing (E/F) domain of PiHR3 (Table 1). Therefore, PiHR3 isa member of a steroid hormone receptor superfamily andclearly assigned to the HR3 subfamily.

3.2. The 20E signalling pathway in G2/M arrest

3.2.1. Effect of RNAi on 20E inducibility of PiEcR-B1,

PiUSP-2, PiHR3 and G2/M arrest

In the IAL-PID2 cell line, it has been reported that the20E-induced G2/M arrest was associated with a largeincrease in the expression level of the PiEcR-B1, PiHR3and PiUSP-2 (Mottier et al., 2004; Siaussat et al., 2005). Toidentify the molecular actors involved in the 20E signallingpathway responsible for G2/M arrest, we inhibited the 20Einducibility of EcR-B1, USP-2, HR3 by RNAi and thenmonitored the effects of each knockdown on the inductionlevel of this cellular response. PiEcR-B1, PiUSP-2, PiHR3dsRNAs at 2 mg/ml combined with LipofectamineTM 2000at 500 ng/ml were incorporated separately into cells. Afterincubation for 10 h, the cells were treated with 10�7M 20Eand the amount of targeted genes expressed was exa-mined by Northern blotting, and the proportion of cells inG2/M phase was evaluated by fluorescence-activated flowcytometry.We first verified that the incorporation of PiHR3,

PiEcR-B1 and PiUSP-2 dsRNAs prevented the 20Einducibility of the corresponding genes. This result wassimilar to that described by Siaussat et al. (2007) in Fig. S1.The IAL-PID2 cells were distributed in the different phasesof cell-cycle as follows: 49(73)%, 23(72)% and 29(74)%in the G0/G1, S, G2/M stages, respectively, and thisdistribution was unchanged with time in normally growingcells (data not shown). In the presence of 20E (10�7M), anincrease in the percentage of cells in G2/M occurred afterexposure to 20E for 12 h and peaked at the end of theexperiment at 68(73)% as compared to 29(74)% in theuntreated cells (Fig. 1A). In combination with PiEcR-B1,PiHR3 and PiUSP-2 dsRNAs, the accumulation of cells inG2/M were greatly reduced (Fig. 1A). Therefore, this resultrevealed the involvement of EcR-B1, USP-2 and HR3 inthe 20E-induced G2/M arrest.For the RNAi controls, we noticed that the incorpora-

tion of PiEcR-B1, PiHR3 and PiUSP-2 dsRNAs hadno effect on the expression of the RpL8 ribosomal gene

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Fig. 1. Correlation between PiEcR-B1, PiUSP-2, PiHR3 and G2/M arrest At 10 h after applying 2 mg/ml PiEcR-B1, PiUSP-2 or PiHR3 dsRNA combined

with Lipofectamine 2000 at 0.5 mg/ml, the cells were treated with 10�7M 20E for various exposure times. At each time, the proportion of cells in G2/M

were analysed by FACS (A). FACS points are means7S.D. (n ¼ 4–7). For RNAi controls, the cells were treated with 2mg/ml RpL8 dsRNA combined

with 0.5 mg/ml Lipofectamine 2000 and the proportion of cells in G2/M were determined by FACS after 8, 18 and 24 h of 20E exposure (B) as well as the

induction level of PiEcR-B1, PiUSP-2 and PHR3 mRNAs was analysed after 8 h of 20E exposure (C).

D. Siaussat et al. / Insect Biochemistry and Molecular Biology 38 (2008) 529–539 533

(data not shown). In addition, the induction of PiHR3,PiEcR-B1 and PiUSP-2 transcripts and the percentage ofIAL-PID2 cells in G2/M were unchanged in the presence ofdsRNA RpL8 (Fig. 1B and C).

3.2.2. Interaction between PiEcR-B1, PiUSP-2 and PIHR3

To examine the interactions between PiEcR-B1, PiUSP-2and PiHR3 in the 20E-induced signalling pathway leadingto G2/M arrest, the IAL-PID2 cells were cultured in thepresence of PiEcR-B1 or PiUSP-2 dsRNA at 2 mg/ml

combined with LipofectamineTM 2000 at 500 ng/ml and theeffect of each transfection on the 20E induction of othergenes was evaluated by Northern blotting. ApplyingPiEcR-B1 dsRNA to the 20E-treated cells resulted in asharp decline in the level of induction of the PiHR3 andPiUSP-2 transcripts (Fig. 2A and C). On the other hand,applying PiUSP-2 dsRNA caused a decrease in the level ofinduction of PiHR3 mRNA (Fig. 2B). Therefore, EcR-B1,USP-2 and HR3 acted in a common 20E signallingpathway responsible for G2/M arrest.

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20E

PiHR3

20E + dsRNA PiEcR-B1

RpL8

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Fig. 2. Relationship between PiEcR-B1, PiUSP-2 and PiHR3. At 10 h

after applying 2 mg/ml PiEcR-B1, PiUSP-2 or PiHR3 dsRNA combined

with Lipofectamine 2000 at 0.5mg/ml, the cells were treated with 10�7M

20E for various exposure times. At each time, the induction level of PiHR3

mRNA was determined by Northern blotting in the presence of PiEcR-B1

dsRNA (A) or PiUSP-2 dsRNA (B) and the induction level of PiUSP-2

mRNA in the presence of PiEcR-B1 dsRNA (C). For Northern blots

controls, a fragment of the cDNA encoding the RpL8 ribosomal protein

of Plodia interpunctella was used.

D. Siaussat et al. / Insect Biochemistry and Molecular Biology 38 (2008) 529–539534

3.2.3. Interaction between B cyclin and G2/M transition

Molecular studies have shown that the 20E-inducedG2/M arrest was preceded by an inhibition of PiCycBexpression (Mottier et al., 2004; Siaussat et al., 2005). Wepursued our functional identification of the 20E signallingpathway in G2/M arrest by demonstrating the role of Bcyclin in the control of G2/M transition in IAL-PID2 cells.Thus, we blocked the expression of PiCycB and thenmonitored the effect of this knockdown on the percentageof cells in G2/M phase. The IAL-PID2 cells were culturedin the presence of LipofectamineTM 2000 at 500 ng/mlcombined with 2 mg/ml dsRNA PiCycB; after incubationfor 10 h, the level of PiCycB expression was analysed byNorthern blotting and the proportion of cells in G2/Mphase was evaluated by fluorescence-activated flowcytometry.

The PiCycB transcript level and the number of cells inG2/M (30(74)%) were unchanged with time in untrans-fected cells (Fig. 3A and B). In contrast, the presence ofdsRNA PiCycB induced both a decline in PiCycBexpression and an accumulation of cells in G2/M thatoccurred from 4 to 24 h with 77(74)% of cells in com-parison with 30(74)% in untransfected cells (Fig. 3A andB). We verified that PiCycB expression was maintained inthe presence of dsRNA RpL8 (Fig. 3C).

3.2.4. Interaction between PiHR3, PiEcR-B1, PiUSP-2 and

B cyclin in inducing G2/M arrest

We blocked the induction of PiEcR-B1, PiUSP-2 andPiHR3, and examined the effect of each disruption on theexpression of PiCycB. As expected, Northern blot analysisrevealed that treatment with 20E alone at 10�7M for 12 hinduced a decrease in the amount of PiCycB transcript(Fig. 4A–C). This inhibitory effect of 20E was dose-dependent and reached 55%, 65% and 80% at concentra-tions of 10�7, 10�6 and 10�5M, respectively (data notshown). Applying 2 mg/ml PiEcR-B1, PiUSP-2, PiHR3dsRNAs to the 20E-treated cells allowed restoration of thelevel of PiCycB expression as compared to untransfectedcells (Fig. 4A–C). We also verified that the inhibition ofPicycB by 20E was maintained in the presence of RpL8dsRNA (Fig. 4D).Finally, we tested the effects of PiCycB silencing

combined with that of PiHR3, PiUSP-2 or PiEcR-B1 onthe 20E-induced G2/M arrest. Fig. 5 shows that theincorporation of PiCycB dsRNA allowed restoration ofG2/M arrest in the presence of PiEcR-B1, PiUSP-2 orPiHR3 dsRNAs. Taken together, these results revealed theexistence of a functional link between EcR-B1, USP-2,HR3 and B cyclin in inducing G2/M arrest.

4. Discussion

In this study, we were interested in dissecting the 20Esignalling pathway responsible for G2/M arrest of imaginalcells using the IAL-PID2 cell line of the lepidopteran P.

interpunctella. Using a 50–30 RACE-based strategy, we firstcloned a 4479 bp cDNA encoding a putative P. inter-

punctella PiHR3 named PiHR3. The deduced amino acidsequence of PiHR3 was highly similar to those of HR3proteins from other lepidopterans, e.g. M. sexta and B.

mori. The highest level of identity was located in the C andE/F domains. The C domain was identical in length (66amino acid residues) with DmHR3, BmHR3, AaHR3 andMsHR3, and has two Cys2–Cys2 zinc finger motifs thatserve as interfaces in both DNA–protein and protein–pro-tein interactions. Using a PiHR3 probe from the C domain,we detected by Northern hybridization one transcript of4.5 kb in 20E-treated IAL-PID2 cells. This result agreeswith previous expression studies performed in vitro inwhich 20E induced a 4.5 kb HR3 mRNA in the CF-203 cellline from Choristoneura fumiferana (Palli et al., 1996), inthe MD-66 cell line from Malacosoma disstria (Palli et al.,

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dsRNA RpL8

PiCycB

0h 24h 0h 24h

4h 8h 12h 18h 24h

0h 8h 12h 18h 24h 0h 8h 12h 18h 24h

Fig. 3. Control of G2/M transition by B cyclin. The IAL-PID2 cells were cultured in presence of Lipofectamine 2000 at 0.5 mg/ml combined with 2 mg/ml

dsRNA PiCycB. After 10 h incubation, the proportion of cells in G2/M phase was evaluated by FACS (A) and in parallel, the level of PiCycB expression

was analysed by Northern blotting (B). For RNAi controls, the cells were treated with 2 mg/ml dsRNA RpL8 combined with Lipofectamine 2000 at 0.5 mg/ml and the level of PiCycB expression was analysed after 24 h incubation (C). For Northern blots controls, a fragment of the cDNA encoding the RpL8

ribosomal protein of Plodia interpunctella was used. FACS points are means7S.D. (n ¼ 3–7).

D. Siaussat et al. / Insect Biochemistry and Molecular Biology 38 (2008) 529–539 535

1995), and in the GV1 cell line from M. sexta (Lan et al.,1997).

We have shown that inhibiting the induction of PiEcR-B1, PiUSP-2 or PiHR3 mRNAs prevented the blockage ofcells in G2/M arrest. This result revealed the involvementof EcR-B1, USP-2 and HR3 in the 20E-induced G2/Marrest. A regulatory relationship between EcR, USP andHR3 has been suggested in other insect cell lines (Lan et al.,1999). Therefore, we examined the interactions betweenthese three molecular actors in the 20E-induced signallingpathway leading to G2/M arrest. A decreased induction ofPiHR3 mRNA was observed by blocking the activation ofthe PiEcR-B1 or the PiUSP-2 gene. On the other hand,applying PiEcR-B1 dsRNA reduced the level of PiUSP-2mRNA induction. Assuming USP-2 and EcR-B1 as theonly isoforms detected in IAL-PID2 cells, USP-2 could bethe single form associated with EcR-B1 in inducing HR3and subsequently G2/M arrest of IAL-PID2 cells. In

contrast, in M. sexta GV1 cells, 20E was able to activatethe expression of the HR3 early gene only when bound tothe EcR-B1-USP-1 heterodimeric complex, and the pre-sence of high levels of USP-2 inhibited the induction ofHR3 mRNA by competing with USP-1 for heterodimer-ization with EcR-B1 (Lan et al., 1999).Activation of the EcR/USP complex by 20E is followed

by the induction of several genes encoding transcriptionfactors such E75, HR3 and HR4 that ultimately regulatethe target genes (Henrich et al., 1999; Andres andThummel, 1992; Thummel, 1995; Thummel et al., 1990;Koelle et al., 1992; Segraves and Hogness, 1990). It wouldbe interesting to test if the inhibitory effect of EcR-B1 andUSP-2 silencing on G2/M arrest can be compensated by anover-expression of PiHR3. This would allow us to demon-strate that PiHR3 induction alone is predominant in therole of EcR-B1 and USP-2 in 20E-regulated G2/M arrest.Nevertheless, some tests have shown that the ectopic

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PiCycB

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18h

24h

0h 4h 8h 12h 18h 24h 0h 4h 8h 12h 18h 24h

20E 20E + dsRNA RpL8

0h 24h 0h 24h

Fig. 4. Interaction between PiEcR-B1, PiUSP-2, PiHR3 and B cyclin. At 10 h after applying 2mg/ml PiEcR-B1, PiUSP-2 or PiHR3 dsRNA combined

with Lipofectamine 2000 at 0.5 mg/ml, the cells were treated with 10�7M 20E for various exposure times. The levels of PiCycB expression in the presence of

PiHR3 (A), PiUSP-2 (B) or PiEcR-B1 (C) were determined by Northern blotting using PiCycB probe. For RNAi controls, the cells were treated with 2 mg/ml RpL8 dsRNA combined with 0.5mg/ml Lipofectamine 2000 and the amount of PiCycB mRNA was evaluated after 24 h of 20E exposure (D). For

Northern blots controls, a fragment of the cDNA encoding the RpL8 ribosomal protein of Plodia interpunctella was used.

D. Siaussat et al. / Insect Biochemistry and Molecular Biology 38 (2008) 529–539536

expression of PiHR3 reaches a level that is much too low tocarry out this experiment. Other assays are in progress tooptimize the production of PiHR3 so that the IAL-PID2cells become better suited to our ectopic expression studies.

The cdk–cyclin complexes are known to modulate theproliferative activity of cells by controlling their progres-sion in different phases of the cell-cycle (Buttitta et al.,2007; Meyer et al., 2000; Boylan and Gruppuso, 2005; Saabet al., 2006). It was reported recently that 20E was able toinduce G2/M arrest of IAL-PID2 cells by inhibiting theexpression of B cyclin (Mottier et al., 2004; Siaussat et al.,2005). To pursue our functional study, we have verifiedthat PiCycB has a role in the control of G2/M transition ofthe IAL-PID2 cell-cycle. We have noticed that inhibitingthe induction of PiEcR-B1, PiUSP-2 or PiHR3 allowedrestoration of the level of PiCycB expression and conse-quently prevented the G2/M arrest of cells. On the otherhand, the silencing of PiCycB combined with that ofPiEcR-B1, PiUSP-2 or PiHR3 induced the accumulation ofcells in G2/M.

Therefore, our RNAi results provided evidence for theinvolvement of EcR-B1, USP-2 and HR3 in a 20E

signalling pathway that induces G2/M arrest of imaginalcells by regulating B cylin expression. It is known that theG2/M arrest of IAL-PID2 cells is followed by a morpho-logical differentiation with the formation of cytoplasmicextensions accompanied by an increased synthesis of btubulin (Siaussat et al., 2007). Other recent RNAiexperiments have demonstrated that the silencing of EcR-B1, USP-2 and HR3 prevented the 20E effects on the btubulin and the shape of IAL-PID2 cells (Siaussat et al.,2007). Taken together, these data revealed the participationof EcR-B1, USP-2 and HR3 in a 20E signalling pathwayinvolved both in G2/M arrest of cells and long term theirmorphological differentiation by modulating the expres-sion level of B cyclin and b tubulin (Fig. 6).In M. sexta, the promoter region of the HR3 gene

contains four putative ecdysone response elements (EcRE)and is activated by 20E through binding of the EcR/USPcomplex to EcRE (Riddiford et al., 2003; Hiruma andRiddiford, 2004). In D. melanogaster, it was demonstratedthat 50-flanking sequences of 20E-regulated target genes arecomposed of a motif homologous to EcRE that appears tobe involved in the level of the ecdysone response (Bruhat

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ycB

) Fig. 5. Effect of the simultaneous silencing of PiEcR-B1, PiUSP-2, PiHR3

and PiCycB on G2/M arrest. At 10 h after applying 2 mg/ml PiEcR-B1,

PiUSP-2 or PiHR3 dsRNA combined with 2mg/ml dsRNA PiCycB, the

cells were treated with 10�7M 20E for 24 h and the proportion of cells in

G2/M phase was evaluated by FACS. FACS points are means7S.D.

(n ¼ 3–6).

20EEcR-B1 USP-2

20E

HR3

B cyclinΒ tubulin

G2/M arrest

Morphological differentiationInduction

Inhibition

Fig. 6. Schematic representation of 20E-induced signalling pathway in the

control of proliferation and differentiation of imaginal wing cells. 20E

binds to a nuclear heterodimeric complex composed of EcR-B1/USP-2

then induces the expression of these two partners and the HR3

transcription factor. This molecular cascade is involved in G2/M arrest

of cells and long term their morphological differentiation by modulating

the expression level of B cyclin and b tubulin (Siaussat et al., 2007).

D. Siaussat et al. / Insect Biochemistry and Molecular Biology 38 (2008) 529–539 537

et al., 1993; Tourmente et al., 1993). To gain more preciseinformation on the relationships between EcR-B1, USP-2,HR3 and B cyclin in the 20E signalling pathway, some

experiments are in progress to characterize the presence ofEcRE and an HR3 response element in the promoterregions of the P. interpunctella HR3 and B cyclin genes,respectively. Some experiments with D. melanogaster andP. interpunctella have revealed that A cyclin acts in synergywith B cyclin to regulate the G2/M transition, and that 20Eis able to modulate the expression of A cyclin in inducingG2/M arrest. It would be interesting to determine whether20E interacts with these two proteins through a commonsignalling pathway.In larvae, the imaginal cells show different develop-

mental responses to 20E in conjunction with the cell-cycle,and the responses depend on the developmental stage andthe concentration to which it is exposed (Koyama et al.,2004; Kawasaki, 1995). In the fourth instar, 20E induces anacceleration of cell proliferation by controlling the G2/Mand G1/S transitions of the cell-cycle at hormonal con-centrations between 1� 10�8M and 5� 10�8M (Koyamaet al., 2004). At the end of the last larval instar, a highproportion of imaginal cells are blocked in G2/M when thehemolymphatic titre of 20E rises above �10�7M (Fain andStevens, 1982; Graves and Schubiger, 1982). This arrestcoincides with the entrance of imaginal cells into the pupaldifferentiation programme leading to the formation ofadult structures. As the IAL-PID2 cell line has beenestablished from P. interpunctella imaginal cells at the endof last larval instar, the participation of EcR-B1, USP-2,HR3 and B cyclin in a common 20E signalling pathway isprobably required in vivo for the 20E-induced G2/M arrestof imaginal cells at the beginning of their pupal differentia-tion. Our functional data are in agreement with the highlevel of USP-2, EcR-B1 and HR3 expression reported forM. sexta imaginal discs in response to the risingecdysteroid titre that precedes pupation (Fujiwara et al.,1995; Jindra et al., 1996).

Appendix A. Supplementary material

Supplementary data associated with this article can befound in the online version at doi:10.1016/j.ibmb.2008.01.001.

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