Effects of juvenile hormone on 20-hydroxyecdysone-inducible EcR ,David Siaussat, Franc¸oise Bozzolan, Isabelle Queguiner, Patrick Porcheron and Ste´phane Debernard Laboratoire de Physiol
Trang 1Effects of juvenile hormone on 20-hydroxyecdysone-inducible EcR ,
David Siaussat, Franc¸oise Bozzolan, Isabelle Queguiner, Patrick Porcheron and Ste´phane Debernard
Laboratoire de Physiologie Cellulaire des Inverte´bre´s, Universite´ Pierre et Marie Curie, Paris, France
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-B1 mRNA induction by 20E was
char-acterized 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 Keywords: differentiation; 20-hydroxyecdysone; imaginal wing cells; juvenile hormone; steroid hormone receptor superfamily
Postembryonic development of insects is characterized by a
growth phase which is punctuated by a series of larval molts
When the larva has attained its characteristic size, the
metamorphic molt(s) is initiated to produce an adult The
larva carries sets of diploid imaginal cells which are tucked
away in its body and contribute little or nothing to the
functioning of the larva [1] The imaginal cells typically
proliferate during the larval life and at metamorphosis
differentiate into new adult organ to replace their larval
counterpart This strategy of early sequestration and
formation of imaginal discs is typical for most imaginal
structures of higher Diptera and for the wing discs of
Lepidoptera [2] This type of development depends on
changes in hemolymphatic levels both of the steroid
hormone 20-hydroxyecdysone (20E) and the sesquiterpe-noid juvenile hormone (JH II)
The contemporary advances in insect endocrinology and tissue culture have led to widespread, even routine use, of organ cultures and cell lines for the investigation of hormonal action [3,4] Nevertheless, most in vitro studies over the ensuing three decades have focused on ecdyster-oids [5,6] while few experiments have been performed for
JH II The first effects of 20E have been reported on lepidopteran and dipteran imaginal discs cultured in vitro [7–9] The mesothoraric wing discs of last larval instar of Plodia interpunctella respond to 20E by an evagination followed by a morphological differentiation and the synthesis of tanned cuticle [8] as described for cultured Drosophila melanogaster discs [9] These diverse 20E-induced responses were inhibited in presence of JH II [10,11] Therefore, these results suggested that in vitro
JH II could counteract the 20E-induced differentiation of imaginals discs but the molecular basis of this action remained largely unknown
Most 20E-induced responses are mediated by a nuclear heterodimeric complex ecdysone receptor (EcR)/ultraspira-cle [12,13] which, when activated by 20E, evokes the sequential transcription of genes encoding proteins that ultimately direct the molt [14–16] These genes were first characterized in D melanogaster and identified as tran-scription factors such as E75 [17], E74 [18], HR3 [19] and BRC [20] In Manduca sexta, some studies have shown that JH II prevented the metamorphic switching of larval tissues such as the epidermis through a modulation of 20E-induced genetic cascade [21,22]
Correspondence to D Siaussat, Laboratoire de Physiologie Cellulaire
des Inverte´bre´s, Universite´ Pierre et Marie Curie, 12 rue Cuvier, 75005
Paris, France Fax: +33 01 44 27 65 93, Tel.: +33 01 44 27 65 09,
E-mail: address: dsiaussat@free.fr
Abbreviations: 20E, 20-hydroxyecdysone; ANS, anisomycin; DIG,
digoxigenin; EcRE, ecdysone response element; JH II, juvenile
hormone; PHR3, Plodia interpunctella hormone receptor 3;
PIE75-B, Plodia interpunctella transcription factor E75-B isoform;
PIEcR-B1, Plodia interpunctella ecdysone receptor-B1 isoform;
UTR, untranslated region.
Database: The nucleotide and amino acid sequence of PIEcR-B1,
PIE75-B, PHR3 are deposited in GenBank under the accession
numbers AY489269, AY566195, AY573570, respectively.
(Received 1 April 2004, revised 13 May 2004, accepted 27 May 2004)
Trang 2Recently, the P interpunctella HR3 and E75
transcrip-tion factors (PHR3, PIE75) were characterized in the
IAL-PID2 cell line established from mesothoracic wing discs
[23,24] PHR3 and PIE75 were identified as components of
a 20E-induced genetic cascade associated with proliferative
arrest, chitin precursor synthesis and long-term
morpholo-gical transformation of IAL-PID2 cells These cellular
events could be referred to as differentiative changes of
imaginal wing cells
This 20E-responsive cell line seemed to be an appropriate
system in which to identify the molecular mechanisms by
which JH II could influence the 20E-induced differentiation
of imaginal wing cells We first tested the sensitivity of
IAL-PID2 cells to JH II examining the effects of this
hormone on 20E-induced late responses such as
prolifera-tive arrest and morphological differentiation Using a 5¢/3¢
RACE/PCR-based strategy, we isolated a cDNA fragment
encoding a putative P interpunctella ecdysone receptor
B1-isoform (PIEcR-B1) Next, we studied the effects of JH II
on 20E-induced genetic cascade reporting the induction
patterns of PIE75-B isoform, PHR3, PIEcR-B1 mRNAs by
20E in the presence of JH II Our results brought evidence
that in vitro JH II prevented 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 PIE75-B, PIEcR-B1 and PHR3 expression
Materials and methods
Cell culture
The IAL-PID2 cell line was established from imaginal wing
discs of final larval instar of P interpunctella Hu¨bner, the
Indian meal-moth [25] The cell line kept its sensitivity to
20E Cells grow as a loosely attached monolayer We
maintained them at 26C in 75-cm2 tissue culture flasks
with 12 mL antibiotic-free Grace’s medium (Gibco BRL)
supplemented with 10% heat-inactivated foetal bovine
serum (Boerhinger Mannheim) and 1% BSA (Calbiochem)
Cells were subcultured weekly to a near confluent
mono-layer Cells were rinsed off the bottom of the flask in a gentle
stream of culture medium and resuspended Cell density
was estimated by counting the cells in an aliquot of the
suspension in a Mallassez haemocytometer under the
microscope All the cultures were initiated by seeding flasks
with 1.5· 106cells JH II and anisomycin (ANS) were from
Scitech (Czech Republic) and Sigma, respectively; 20E was a
gift from R Lafont (UPMC, Paris, France) Stock solutions
of JH II were prepared in dimethyl sulfoxide (DMSO) and
were stored at )20 C in glass vials coated with 1%
polyethylene glycol 20 000 to decrease possible adsorptive
loss [26] All media containing JH II were just prepared
before culture, then they were sonicated and thoroughly
vortexed briefly For use in culture, 20E and ANS dissolved
in ethanol and JH II in dimethyl sulfoxide were diluted in
appropriate volumes of sterile Grace’s medium
supplemen-ted with foetal bovine serum and BSA Then, these solutions
were added directly to cell cultures by using glass capillary
pipettes Final ethanol and dimethly sulfoxide
concentra-tions in all treatments and control cultures were maintained
at less than 0.1% to prevent any toxic effect of the solvent
JH II becomes insoluble in aqueous solution above
2· 10)5M [27], therefore the highest concentration used was 10)6M
Isolation of RNA and cDNA synthesis Total RNAs from cells were extracted with TRIzol reagent (Gibco, BRL) and quantified by spectrophotometry at
260 nm The quality of RNA was checked by electrophor-esis on a formaldehyde/agarose gel (1%) Using the first strand synthesis kit (Roche), 1 lg total RNA was reverse transcribed into single-stranded cDNA with AMV reverse transcriptase and Oligo-p(T)15 as primer For 5¢- and 3¢-RACE, cDNA was synthesized from 1 lg total RNA
at 42C for 1.5 h using the SMART RACE cDNA Amplification kit (Clontech) with 200 U of Superscript II (GibcoBRL), 5¢- or 3¢- CDS-primer and SMART II oligonucleotide, according to the instructions in the kit PCR amplification and cloning
Two degenerate DNA primers (ED1, ER1) were designed
on the basis of conserved amino acid sequences (KCQECRL and VEFAKGL) from the DNA and ligand binding regions of D melanogaster, Bombyx mori, Tenebrio molitor, Choristoneura fumiferana and M sexta ecdysone receptors (EcRs) PCR was carried out in 100 lL final volume including 10 mM KCl, 6 mM ammonium sulfate,
20 mM Tris/HCl, pH 8, 2.5 mM MgCl2 with 2.5 U High Expand Fidelity DNA polymerase (Boerhinger Mannheim) and 25% of the cDNA produced by reverse transcription of the total RNAs The degenerate primers ED1 5¢-forward primer (5¢-AARTGYCARGARTGYMGNYT-3¢), ER1 5¢-reverse primer (5¢-CARNCCYTTNGCRAAYTCNAC-3¢)
at 1 lM and each dNTP at 0.8 mM were then added Following an initial 5 min denaturation at 94C, the thermal amplification procedure included 5 cycles of denaturation 1 min at 94C, annealing at 55 C for
1 min and an elongation at 72C for 1 min The reaction was repeated for 30 cycles with an annealing temperature of
45C
The blunt-ended PCR product was purified by agarose gel electrophoresis and cloned with Stratagene’s pCR-ScriptTMSK(+) cloning kit following the manufac-turer’s instructions After colony isolation, DNA minipreps were prepared and correct insertion was determined by restriction enzyme analysis The DNA clone containing the proper insert was sequenced by the dideoxy chain termin-ation method [28] (Genome Express, Grenoble, France) One 477-bp RT/PCR product was isolated and sequenced
Rapid amplification of cDNA 5¢/3¢-terminal ends (5¢/3¢-RACE)
The 5¢- and 3¢-regions of the corresponding cDNA were obtained by 5¢- and 3¢-RACE (SMART RACE cDNA amplification kit) following the manufacturer’s instructions For 5¢-RACE, we used 2 lL of 5¢-RACE-ready cDNA with
a specific reverse primer 5¢-Race PIX (5¢-CCTGGCG GCCTCTGGTGGTGGCGG-3¢) and Universal primer Mix (UPM, Clontech) as the forward anchor primer The 3¢-RACE amplification was carried out with UPM
as the reverse primer and a specific forward primer
Trang 33¢-Race PIY (5-¢GCGGGGCTCGTGTGGTACCAG
GACG-3¢) Touchdown PCR was performed using hot
start as follows: after 1 min at 94C, five cycles of 30 s
at 94C and 5 min at 72 C, then five cycles of 30 s at
94C, 30 s at 70 C and 3 min at 72 C, then 25 cycles
of 30 s at 94C, 30 s at 68 C and 5 min at 72 C, then
7 min at 72C The PCR products were purified and
cloned as described above By merging the overlapping
sequences obtained from the 5¢- and 3¢-RACE, a 6081-bp
cDNA fragment was generated and named PIEcR
Generation of DIG -labelled probe
PIEcR cDNA was digoxigenin (DIG)-labelled by PCR
using the PCR DIG probe synthesis kit (Roche) with a
pair of specific primers CED 5¢-forward primer
(5¢-CGCTGGTCCAACAACGGAGGG-3¢), CER 5¢-reverse
primer (5¢-TGCCGGTGACAACTCCTCACG-3¢) The
DIG-labelled probe was used at a concentration of
25 ngÆmL)1in hybridization solution
Northern blotting
Northern blot hybridization analysis was performed
according to the manufacturer’s instructions RNA
sam-ples (15 lg) were denatured with formamide (50%) and
formaldehyde (2.2M), separated on 1% denaturating
agarose gel and transferred to a Boerhinger Mannheim
positively charged nylon membrane Blotted RNA was
hybridized overnight at 55C with the PIEcR-B1 probe,
at 42C with PHR3 probe and at 45 C with PIE75-B
specific probe located in the N-terminal region of A/B
domain A DIG-labelled fragment of the cDNA encoding
the RpL8 ribosomal protein of P interpunctella was used
as control probe An immunological signal detection by
cheluminescence was performed as described in Roche’s
DIG system User’s Guide for filter hybridization A
molecular RNA marker ladder DIG-labelled (Roche) was
run in parallel to determine the molecular mass of
bybridizing RNAs
Results
Effects of JH II on 20E-induced late responses
in IAL-PID2 cells
We first tested the sensitivity of cells to JH II by studying the
effects of this hormone on the 20E-induced late responses
such as proliferative arrest and morphological
differenti-ation of IAL-PID2 cells
Proliferative arrest The IAL-PID2 cells were seeded at
1.5· 106 per flask and cultured under normal growth
conditions for 36 h, in our model this period of time
corresponded to the population doubling time [29,30] Cells
were then treated with only 20E at 10)7Mor in combination
with JH II at various concentrations for 36 h At the end of
treatment, the cell density was evaluated Fig 1 indicates
that 20E alone induced a striking decrease of cell
prolifer-ation By contrast, in combination with JH II at 10)6or
10)7M, cells grew at almost the normal rate Intermediate
levels of cell proliferation were attained at 5· 10)8, 10)8
and 10)9M JH II We checked that 0.1% ethanol or dimethly sulfoxide or JH II at 10)6Malone had no effect on cell growth (Fig 1)
Morphological changes After 48 h of 20E treatment at
10)7M, the treated cultures (Fig 2C) appeared to be much less dense than control cultures (Fig 2A) The cells were elongated and aggregated, often producing long processes which formed connections between different aggregates (Fig 2C) In combination with JH II at 5· 10)8, 10)8and
10)9M, the cultures were always composed of pseudo-epithelial aggregate structures (Fig 2F, G and H) How-ever, we noted an increase in the size of aggregates that was related to an increase in cell density as compared to cultures treated by 20E alone (Fig 2C) At the highest concentra-tions of JH II (10)6, 10)7M), the cultures did not show any cell aggregation, or cell cytoplasmic extensions and the cell density was slightly lower than in the control cultures (Fig 2D and E) In the presence of JH II alone at 10)6M, the shape and the distribution of the cells in culture were similar to those of control cultures (Fig 2B) These results showed that JH II was able to inhibit efficiently the effects
of 20E both on cell proliferation and morphological changes
of IAL-PID2 cells
Isolation and characterization ofP interpunctella EcR-B1 mRNA
Cloning of a PIEcR cDNA frament We wondered whether the inhibitory effect of JH II could imply an action
of this hormone on molecular events which occur very early
in the cellular response to 20E Therefore, we examined the effects of JH II on the 20E-induced genetic cascade and decided to clone a P interpunctella ecdysone receptor Using a 5¢/3¢-RACE/PCR-based strategy, a 6081-bp cDNA fragment was generated and named PIEcR (Fig 3) The 3¢-untranslated region (3¢UTR) is long (4074 bp) and two putative polyadenylation signals are present (Fig 3) The
Fig 1 Effect of 20E and JH II on the proliferation of IAL-PID2 cells The IAL-PID2 cells were seeded at 1.5 · 10 6 per flask and cultured under normal growth conditions for 36 h Cells were then grown for
36 h in Grace’s medium containing no hormone or only 20E at 10)7M
or in combination with JH II at various concentrations At the end of treatment, the cell density was evaluated.
Trang 4ORF which starts from AUG consistent with the
transla-tion start consensus sequences among general eukaryotes
[31] and D melanogaster [32] encodes 541 amino acids,
predicting a 62-kDa protein This ORF includes five
domains (A/B, C, D, E, F) that are characteristic members
of the steroid hormone nuclear receptor superfamily
(Fig 3)
Sequence comparison A high degree of amino acid identity with C fumiferana EcR (CfEcR) [33], M sexta EcR (MsEcR) [34,35], B mori EcR (BmEcR) [36,37],
T molitor EcR (TmEcR) [38] and D melanogaster EcR (DmEcR) [12,39] was observed in both the DNA binding region (C region) and the ligand binding domain (E region)
of PIEcR (Table 1) Therefore, PIEcR was a member of the
Fig 2 Effect of 20E and JH II on the morphology of IAL-PID2 cells IAL-PID2 cells were grown for 48 h in Grace’s medium containing 0.1% ethanol (A) or 10)6M JH II (B) or 10)7M 20E (C) or 10)7M 20E in combination with JH II at various concentrations 10)6M (D), 10)7M (E),
5 · 10)8M (F), 10)8M (G) and 10)9M (H) Each panel shows the representative area of three replicates The bar in A represents 40 lm in A, B, C,
D, E, F, G and H.
Trang 5steroid hormone nuclear receptor superfamily and was
clearly assigned to the EcR subfamily
EcR exists in different isoforms) EcR-A, EcR-B1 and
EcR-B2 [39] All three share common DNA- and
ligand-binding domains, but each has its own isoform-specific
segment in the N-terminal region of A/B domain which
contains a transactivating domain [40] The predicted
sequence of A/B domain of PIEcR exhibited significant
amino acid identities with the corresponding region of B1
isoform of other insect EcRs, despite differences in the
domain length (Fig 4) Overall in the A/B region, there was
91, 87, 82, 52, and 42% amino acid identity based on the
Plodiasequence with CfEcR-B1, BmEcR-B1, MsEcR-B1,
DmEcR-B1, and TmEcR-B1, respectively (Fig 4) The
strongest similarities were confined to the two ends of the B1
isoform specific segment in the N-terminal region (Fig 4)
There was no similarity to the N-terminal specific regions of either the A or the B2 isoform (data not shown) All of these results indicate that PIEcR is a B1 type isoform
Effect of 20E and anisomycin on induction of PIEcR-B1 mRNA Using a PIEcR-B1 specific probe located in the N-terminal region of A/B domain, the Northern blot hybridization on total RNAs revealed a 6-kb transcript whose expression level was higher in presence of 20E The size of this transcript is in agreement with the length of the corresponding cDNA This 20E-induced transcript could thus encode a putative P interpunctella ecdysone receptor-B1 isoform (PIEcR-receptor-B1) To analyse how the expression of PIEcR-B1 is regulated by 20E, IAL-PID2 cells were cultured in Grace’s medium containing 20E at 10)7M for different continuous time exposures In the absence of 20E,
Fig 3 Nucleotide and deduced amino acid
sequences of PIEcR Nucleotide numbers are
given on the left and the amino acid numbers
on the right Letters in the right margin
des-ignate domains The DNA binding domain
(C region) is underlined and the ligand binding
domain (E region) is underlined with dashes.
The helix–turn–zipper motif is
double-under-lined and two polyadenylation signals in the
3¢UTR are designed in bold type Degenerate
primers (ED1) and (ER1) (shown in bold type)
were used to generate a cDNA fragment of
477 bp by RT/PCR The PIX and PIY
pri-mers used for the 5¢/3¢)RACE are shown in
italic and bold type The PIEcR-B1 specific
probe was generated by PCR with the two
primers, CED and CER, shown in italic type.
Trang 6PIEcR-B1 was constitutively expressed at low level over
time (data not shown) By contrast, the pattern of
PIEcR-B1 mRNA induction by 20E was characterized by a
biphasic response with peaks at 2 h and 18 h (Fig 5A) To
define the minimal concentration of 20E required for an
induction of PIEcR-B1 mRNA, IAL-PID2 cells were
exposed to various concentrations of 20E for 18 h As
shown in Fig 5B, a significant induction of PIEcR-B1
mRNA was first observed at 10)7M20E with an increase up
to 10)5M
To determine whether 20E directly initiated the
tran-scription of PIEcR-B1, we studied the effects of protein
synthesis inhibitor, ANS on the induction of PIEcR-B1
mRNA The IAL-PID2 cells were cultured in Grace’s
medium containing 20E (10)7M) with ANS (5 lgÆmL)1) for
different continuous time exposures Under these culture
conditions, the presence of ANS caused 94% inhibition of
protein synthesis (n¼ 4) and the cells remained viable even
24 h after ANS removal (data not shown) The Fig 6 shows
that ANS caused a slight reduction in the level of PIEcR-B1
mRNA within the first 2 h but neither completely prevented the initial increase induced by 20E This observation suggested that the majority of the induction of PIEcR-B1 mRNA by 20E was independent from protein synthesis and thus probably due to direct action of 20E on the PIEcR-B1 gene The most surprising result was that the observed declines in the level of PIEcR-B1 mRNA did not occur in the presence of ANS, suggesting that a 20E-induced protein(s) synthesis was involved in these decreases
Regulation of 20E-inducedPIEcR-B1, PIE75-B, PHR3 transcripts by JH II
The Plodia HR3 and E75 transcription factors have been identified recently as putative actors of a 20E-induced genetic cascade leading to the inhibition of cell proliferation and long-term morphological changes of IAL-PID2 cells [23,24,41] To examine the effects of JH II on this genetic cascade, IAL-PID2 cells were cultured in Grace’s medium containing both 20E at 10)7M and JH II at 10)7M for different continuous time exposures The induction patterns
of PIE75-B, PHR3, PIEcR-B1 mRNAs were determined under these experimental conditions
We remarked that in presence of JH II alone at 10)7Mor
in absence of hormone, PIE75-B and PIEcR-B1 were constitutively expressed at a low level over time (Fig 7A and C) whereas PHR3 mRNA was never detectable (Fig 7B) In the presence of 20E alone, PHR3 mRNA was detectable at 2 h, reached a maximum by 8 h and then declined (Fig 7B) whereas PIE75-B mRNA was already highly induced after 1 h, rapidly disappeared by 2 h, then peaked again at 8 h and was maintained at a high level (Fig 7C) In combination with JH II, PHR3 and PIE75-B transcripts showed temporal patterns similar to those obtained in response to 20E alone Furthermore, the presence of JH II induced an increase in induction level of these two transcripts (Fig 7B and C) We also noticed that the overexpression of PIE75-B occurred only within the first
4 h whereas that of PHR3 was maintained during the 32-h culture period As concerns PIEcR-B1, JH II had no effect
Table 1 Comparison of amino acid sequences of C and E regions
between PIEcR and homologs D melanogaster EcR (DmEcR [12]),
B mori EcR (BmEcR [37]), M sexta EcR (MsEcR [35]), C fumiferana
EcR (CfEcR- [33]), and T molitor EcR (TmEcR [38]) Indicated are the
lengths of C and E regions of the EcR nuclear receptors (number of
amino acids) and the identity vs PIEcR expressed as percentage of the
PIEcR sequence.
C region E region
Identity
(%)
Length (amino acids)
Identity (%)
Length (amino acids) PiECR 100 66 100 222
BmEcR 98 66 87 218
CfEcR 98 66 88 222
MsEcR 98 66 91 222
TmEcR 88 66 66 218
DmEcR 94 66 71 220
Fig 4 Alignment of the amino acid sequence of A/B region of PIEcR with D melanogaster EcR-B1 (DmEcR-B1 [12]), B mori EcR-B1 (BmEcR-B1 [37]), M sexta EcR-B1 (MsEcR-B1 [35]), C fumiferana EcR-B1 (CfEcR-B1 [33]), and T molitor EcR-B1 (TmEcR-B1 [38]) Gaps are introduced to optimize alignment Asterisks indicate identical residues and dots indicate conservative substitutions Multiple sequence alignment was performed using [58].
Trang 7on the initial 20E-induced increase of PIEcR-B1 mRNA
whereas it prevented the second increase (Fig 7A)
To determine the effectiveness of JH II, we cultured
IAL-PID2 cells with 10)7M20E alone and in combination
with JH II at various concentrations Based on the times
required for the maximum induction of mRNAs, the level of
PIE75-B, PHR3 and PIEcR-B1 mRNAs was assessed after
1, 8 and 18 h exposure, respectively Fig 8B and C show
that after 1 h and 8 h exposure to 20E, the amount of
accumulated PIE75-B and PHR3 mRNAs increased in
parallel with concentration of JH II up to 10)6M Thus, the
suppressive effect of JH II on the second 20E-induced
increase in PIEcR-B1 mRNA was also
concentration-dependent when assayed after 18 h exposure to 20E
(Fig 8A) These results indicated that the effects of JH II
on PHR3, PIEcR-B1, PIE75-B mRNAs induction by 20E
were both dependent on the amount of JH II present and
significant from 10)8MJH II
Finally, all of these results demonstrated that JH II acts
on the 20E-induced genetic cascade by differential modu-lating of the expression of PIEcR-B1, PIE75-B and PHR3
Discussion
Our main objective was to identify some specific molecular mechanisms through which in vitro JH II was able to
Fig 6 Effect of anisomycin on PIEcR-B1 mRNA induction Fifteen
micrograms of total RNAs from IAL-PID2 cells cultured in Grace’s
medium with 20E at 10)7M or with 10)7M 20E and 5 lgÆmL)1
aniso-mycin for various times of exposure were analysed by Northern blots
and hybridized with PIEcR-B1 probe mRNA levels of PIEcR-B1 are
shown as percentage of its mRNA level in IAL-PID2 cells cultured with
10)7M 20E for 18 h Points are means ± SD (n ¼ 5–11).
Fig 7 Effect of 20E and JH II on induction of PIE75-B, PHR3 and PIEcR-B1 mRNA Fifteen micrograms of total RNAs from IAL-PID2 cells cultured in Grace’s medium with 20E at 10)7M alone or in combination with 10)7M JH II for various times were analysed by Northern blots hybridized with PIEcR-B1 (A), PHR3 (B) or PIE75-B (C) probes Levels of the mRNAs of PIE75-B, PHR3 and PIEcR-B1 are shown as percentages of their respective mRNA levels in IAL-PID2 cells cultured with 10)7M 20E for 1 h, 8 h and 18 h Points are means ± SD (n ¼ 5–14).
Fig 5 Induction of PIEcR-B1 mRNA by 20E Fifteen micrograms of
total RNAs from IAL-PID2 cells cultured in Grace’s medium with
20E at 10)7M for various times of exposure (A) or with 20E for 18 h at
different concentrations (B) were separated on agarose (1%)
formal-dehyde gel, transferred to nylon membrane and hybridized with
PIEcR-B1 probe A fragment of the cDNA encoding the RpL8
ribo-somal protein of P interpuntella was used as control probe.
Trang 8counteract 20E-induced differentiation of imaginal discs To
accomplish this work, we used a 20E responsive IAL-PID2
cell line established from mesothoracic imaginal wing discs
isolated at the last larval instar of P interpunctella
lepidop-tera [25] These imaginal cells respond to 20E at 10)7Mby
an arrest of cell proliferation and long-term morphological
changes marked by the formation of pseudoepithelial
aggregates structures These 20E-induced late responses of
IAL-PID2 cells resemble, in general terms, the metamorphic
transformation of many different imaginal cell types in
D melanogasterand other holometabolous insects [42,43]
A 10)7M concentration of 20E was both close to
physio-logical levels of 20E (in the order of 10)7to 10)6M[44] or
2· 10)8to 6· 10)6M[45]) and sufficient to induce in vitro
eversion and differentiation of imaginal discs [9,46] First,
we tested the sensitivity of IAL-PID2 cells to JH II and showed that in combination with 20E, JH II inhibited significantly the 20E-induced late responses from 10)8Mas already reported in D melanogaster Kc cells [44] This concentration of JH II was close to physiological levels which were estimated at 4· 10)9to 2· 10)7M[45]
To examine the effects of JH II on the 20E-induced genetic cascade, we first cloned a 6081-bp cDNA encoding a putative P interpunctella ecdysone receptor named PIEcR The deduced amino acid sequence of PIEcR was most highly similar to those of EcR proteins from other lepidopterans, M sexta [34,35], C fumiferana [33] and
B mori[36,37] The highest identity was located in the C and E domains The C domain was identical in length (66 amino acids) to DmEcR, CfEcR, MsEcR, TmEcR, BmEcR and has two Cys2–Cys2 type zinc finger motifs that serve as interfaces in both DNA–protein and protein–protein inter-actions [47] The E domain is known to be involved in ligand binding, transcriptional activation (or repression), nuclear translocation and dimerization [48] It has been demonstra-ted that EcR needs to form a heterodimer with the ultraspiracle protein for binding to the EcRE sequence and transactivation [13] The helix–turn–zipper motif which seems to be essential for receptor dimerization [49] is present
in PIEcR (Fig 3)
PiEcR had significant amino acid identities (especially with the B1 isoform) of other insect EcRs and a strong degree of identity was confined to the two ends of N-terminal region of the A/B domain Using a B1 isoform-specific probe from the A/B region of PIEcR, we detected by Northern hybridization one transcript of 6 kb, close in size to those of DmEcR-B1 (6.8 kb), MsEcR-B1 (6 kb), CfEcR-B1 (6 kb), BmEcR-B1 (6.2 kb) and TmEcR-B1(6.5 kb) mRNAs This result revealed the expression of ecdysone receptor B1 isoform in imaginal wing cells of
P interpunctellaat the last larval instar
In the fifth larval instar of M sexta, it has been reported
in vitrothat 20E induced a coexpression of MsEcR-B1 and MsEcR-A during the metamorphic switching of abdominal epidermis The two isoforms were directly upregulated by 20E but differed in their responsiveness to 20E and to protein synthesis inhibitors [34] The pattern of PIEcR-B1 mRNA induction by 20E showed a biphasic response which was similar to that of MsEcR-B1 mRNA Inhibition of protein synthesis slowed the rapid accumulation of PIEcR-B1 mRNA and prevented its subsequent decline This result agrees with the effects of anisomycin on the induction of MsEcR-B1mRNA by 20E Therefore, during the differen-tiation of imaginal wing cells, the expression of PIEcR-B1 was regulated both by 20E and 20E-induced protein(s), presumably transcription factors in the same manner as MsEcR-B1 at the time of metamorphic switching of abdominal epidermis
Some developmental studies have shown that EcR isoforms are expressed in a tissue- and stage-specific manner, thus contributing to the spatial and temporal diversity of the response to 20E [33,34,37–39,50,51] Our study revealed that EcR-B1 seemed to be the single form associated with 20E-induced morphological changes of imaginal wing cells of Plodia Using a probe common to all EcR isoforms, we succeeded to detect a second 20E-inducible transcript whose expression level was much lower
Fig 8 Concentration–response curves for the effectiveness of JH II.
IAL-PID2 cells cultured in Grace’s medium with 10)7M 20E alone or
in combination with JH II at various concentrations and the levels of
expression of PIE75-B, PHR3 and PIEcR-B1 were assessed by
Nor-thern blotting after 1, 8 and 18 h exposure, respectively mRNA levels
of PIE75-B, PHR3 and PIEcR-B1 are shown as percentages of their
respective mRNA levels in IAL-PID2 cells cultured with 10)7M 20E
for 1, 8 and 18 h Points are means ± SD (n ¼ 7–13).
Trang 9than that of PIEcR-B1 mRNA (data not shown) If this
transcript is the Plodia EcR-A isoform, then Plodia imaginal
discs would be similar to those of Manduca during the pupal
predifferentiative phase necessary for eversion and cuticle
synthesis [34]
In several holometabolous insects, at the end of last instar
larvae, imaginal discs are characterized by a high proportion
of cells blocked in the G2phase in response to the rising
ecdysteroid titre prior to pupation [52,53] In IAL-PID2
imaginal cells, recent works have shown that the G2arrest is
associated with high induction of PHR3 mRNA and a
decrease in the expression level of A and B cyclins which
occurred after 8 h of 20E continuous treatment [41] We
noticed that the induction of PIEcR-B1, PIE75-B, PHR3
mRNAs by 20E was enhanced as early as 2 h of 20E
exposure and thus prior to the inhibition of A and B cyclin
expression According to all these data, we suggest that 20E
could initiate a genetic cascade involving EcR-B1, HR3,
E75-B to regulate the expression of cyclins and ultimately
the G2/M transition Some RNA interference experiments
are in progress to identify the sequence of the molecular
events linking the 20E action with proliferative arrest It has
been reported that in combination with 20E, JH II was able
to increase the induction level of PHR3 mRNA, restore the
expression of A and B cyclins and consequently prevent G2
arrest [41] Our study confirmed the effect of JH II on the
20E inducibility of PHR3 and revealed that this hormone
also modulated the induction level of PIEcR-B1 and
PIE75-B mRNAs This action of JH II provided an argument
for the existence of a strong correlation between the
20E-induced genetic cascade, cyclins and proliferative arrest
JH II had no effect on the initial 20E-induced increase in
PIEcR-B1mRNA whereas it prevented the second increase
This result was agreement with that obtained on EcR
homologous gene in the M sexta epidermis In this tissue,
JH II prevented the 20E-induced metamorphic switching by
regulating the induction of EcR by 20E [21,22] On the other
hand, our study demonstrated that JH II increased the level
of PIE75-B without modifying its induction pattern by 20E
This JH II effect was similar to that reported on the 20E
inducibility of the E75-A isoform in the cultured silk gland
of Galleria mellonella and in M sexta epidermis [21,22,54]
The JH II effects on 20E-induced PHR3, PIE75-B,
PIEcR-B1mRNAs were concentration dependent and significant
at 10)8M This JH II concentration was identical to that
found in the hemolymph at the onset of the fifth larval molt
of M sexta [55]
Molecular data from Manduca wing discs have
demon-strated that BR-C transcription factor plays a key role for
their differentiation and that its expression is clearly
controlled by JH II [56] Therefore, in order to complete
our work, some experiments are in progress to characterize
a Plodia BR-C and then to examine the effects of JH II
on its induction pattern by 20E in our IAL-PID2 imaginal
wing cells
The increased amounts of both PIE75-B and PHR3
mRNAs by JH II were most probably due to an increased
transcription rate One possible action of JH II is to stabilize
the open chromatin structure of the PIE75-B and PHR3
promoters around the ecdysone response element (EcRE) so
that 20E can readily access the binding site of EcR and thus
induce an increase in transcription level Our studies,
however, have not ruled out a possible additional effect of
JH II on increasing the stability of HR3 and E75-B mRNAs
We noticed that JH II had no early effect on the response
of PIEcR-B1 while it regulated quantitatively the level of PIE75-Band PHR3 mRNAs induced by 20E These results suggested that the later effect of JH II on the expression pattern of PIEcR-B1 could be due to differences in induction level of PIE75-B and PHR3 mRNAs by 20E in the presence of JH II or to some other factors not yet identified It has been suggested that HR3 genes are candidates for the feedback repression of EcR [57] In
M sexta and D melanogaster, some studies have shown that DmEcR and MsEcR were repressed when DmHR3 and MsHR3 begun to be highly expressed [33,51] Such a correlation was found in our IAL-PID2 cells for the expression of PIEcR-B1 and PHR3 Therefore, in the presence of JH II, the increased accumulation of PHR3 mRNA could inhibit the second increase in PIEcR-B1 mRNA and block the 20E-induced molecular cascade leading to proliferative arrest and morphological differen-tiation of IAL-PID2 cells This second rise in EcR-B1 mRNA seen in response to 10)7M 20E in vitro is probably required for the differentiative cellular changes
of lepidopteran wing discs
Finally, we demonstrated that in vitro JH II was able to prevent the 20E-induced differentiation of imaginal wing cells In addition, for the first time, our study revealed that
JH II also modulates differently the 20E inducibility of EcR-B1 and E75-B isoforms in imaginal cells This JH II effect on 20E-induced genetic cascade could be associated with its action in prevention of differentiative program of imaginal wing discs at the onset of metamorphosis
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