Báo cáo khoa học: Insect cytokine growth-blocking peptide signaling cascades regulate two separate groups of target genes docx

Insect cytokine growth-blocking peptide signaling
cascades regulate two separate groups of target genes
Yosuke Ninomiya
1
, Maiko Kurakake
2
, Yasunori Oda
2
, Seiji Tsuzuki
2
and Yoichi Hayakawa
2
1 Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan
2 Department of Applied Biological Sciences, Saga University, Japan
In animals, tyrosine hydroxylase (TH, EC 1.14.16.2)
and 3,4-dihydroxy-l-phenylalanine (Dopa) decarboxyl-
ase (DDC, EC 4.1.1.26) are required for the produc-
tion of Dopa and dopamine [1,2]. Because Dopa is
required for tanning of newly formed cuticle and dopa-
mine has been shown to exert many neurohormonal
functions in insects, both enzymes are essential for the
maintenance of life [3–6]. Furthermore, Dopa and
dopamine are also oxidized by a system of phenoloxid-
ases and cofactors to form melanins that produce the
various black and brown patterns of the cuticles in a
broad range of insect species [7,8].
We previously demonstrated that transcription lev-
els as well as enzyme activities of TH and DDC in
larval cuticles are elevated during molt periods in
the armyworm, Pseudaletia separata [9,10]. Morpho-
logical analysis showed preferential distribution of

both enzymes in the epidermal cells beneath the
black stripes in the dorsal surface of armyworm lar-
vae. Because the black stripes become much darker
and wider after each larval ecdysis, both enzymes
are thought to contribute to production of melanins
during molts [9]. Furthermore, the periodic
expression of both enzyme genes was found to be
Keywords
calcium ion; Dopa decarboxylase;
extracellular signal-regulated kinase; growth-
blocking peptide; tyrosine hydroxylase
Correspondence
Y. Hayakawa, Department of Applied
Biological Science, Saga University, Honjo-1,
Saga 840-8502, Japan
Fax ⁄ Tel: 81 952 28 8747
E-mail:
(Received 9 October 2007, revised 4
December 2007, accepted 18 December
2007)
doi:10.1111/j.1742-4658.2008.06252.x
Growth-blocking peptide (GBP) is a 25 amino acid insect cytokine found
in lepidopteran insects that has diverse biological activities, such as larval
growth regulation, paralysis induction, cell proliferation, and stimulation
of immune cells. GBP also enhances expression of the tyrosine hydroxylase
(TH, EC 1.14.16.2) and 3,4-dihydroxy-l-phenylalanine (Dopa) decarboxyl-
ase (DDC, EC 4.1.1.26) genes, which elevate dopamine levels in insect epi-
dermal cells. We used insect epidermis and cultured cells to define the role
of the GBP signaling pathway in the enhancement of TH and DDC gene
expression. It has been recently reported that robust expression of the

DDC gene requires activation of extracellular signal-regulated kinase
(ERK) in epidermal cells of wounded Drosophila embryos. This study con-
firmed that GBP activates ERK, but this activation is not directly linked to
the enhancement of TH and DDC gene expression. One of the GBP path-
way components is phospholipase C, whose activation is essential for the
activation of ERK and elevation of expression of both enzyme genes. The
downstream signaling pathways diverge to ERK activation through acti-
vated protein kinase C and expression of the enzyme genes through inositol
triphosphate receptor-mediated Ca
2+
influx from extracellular fluid. Our
data indicate that the diverged GBP signaling pathways enable GBP to
exert completely different biological functions, even in a single cell type.
Abbreviations
DAG, diacylglycerol; DDC, Dopa decarboxylase (EC 4.1.1.26); Dopa, 3,4-dihydroxy-
L-phenylalanine; ERK, extracellular signal-regulated kinase;
GBP, growth-blocking peptide; IP
3
, inositol triphosphate; MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase kinase;
PKC, protein kinase C; PLC, phospholipase C; PTTH, prothoracicotropic hormone; TH, tyrosine hydroxylase (EC 1.14.16.2).
894 FEBS Journal 275 (2008) 894–902 ª 2008 The Authors Journal compilation ª 2008 FEBS
regulated by the insect cytokine, growth-blocking
peptide (GBP) [10].
GBP was initially identified as the factor responsible
for the reduced growth exhibited by P. separata larvae
after parasitization by the parasitic wasp Cotesia kar-
iyai [11–13]. Analysis of the mechanism by which GBP
retards larval growth revealed that GBP activates
DDC activities in epidermal cells and elevates dopa-
mine concentrations in the integuments [14–16]. Subse-

quent studies provided evidence that part of the
increased dopamine in the integuments is released into
the hemolymph, and consequently retards the normal
development of armyworm larvae [15]. Furthermore,
recent studies showed that the activation of DDC is
induced by its transcriptional enhancement through
GBP-induced elevation of cytoplasmic Ca
2+
concen-
trations in the epidermal cells [10]. Therefore, we spec-
ulated that the increased level of cytoplasmic Ca
2+
affects a certain transcription enhancer that stimulates
expression of the DDC gene. A previous study also
suggested the involvement of mitogen-activated protein
(MAP) kinases in epidermal DDC expression: in
wounded Drosophila embryos, activation of extracellu-
lar signal-regulated kinase (ERK) is required for the
activation of DDC gene expression near wounded sites
in the epidermal integument [17].
In this study, we characterized the signal transduc-
tion pathway of GBP to clarify whether GBP can acti-
vate ERK and whether its activation is required for
the expression of TH and DDC genes. Using insect
integuments and cultured cells, we demonstrated that
GBP activates ERK but that its activation is not nec-
essary for the induction of expression of either enzyme
gene.
Results
GBP-induced activation of ERK in integuments

Prior studies showed that expression of TH and DDC
genes was enhanced in integuments isolated from
armyworm larvae when they were incubated with GBP
[10]. We examined whether GBP induces dual phos-
phorylation (activation) of ERK in the larval integu-
ments under the same conditions. As shown in
Fig. 1A, dual phosphorylation of ERK was clearly
enhanced when the integuments were incubated with
10 nm GBP for 5 min, but this phosphorylation was
completely blocked by U0126, a MAP kinase kinase
(MEK) inhibitor that reduces the phosphorylation and
activation of the ERK–MAP kinase. In contrast, addi-
tion of U0126 to the incubation medium did not pre-
vent GBP-induced expression of TH and DDC genes
(Fig. 1B), thus indicating that activated ERK is not
required for GBP-induced gene expression of either
enzyme in epidermal integuments isolated from army-
worm larvae.
GBP-induced ERK activation in MaBr4 cells
For the detailed characterization of transcriptional reg-
ulatory inputs involved in GBP-dependent activation
of expression of TH and DDC genes, we used cultured
dpERK
ERK
Relative ERK activity and
total ERK amount
TH
DDC
Actin
4

3
2
1
0
*
0nM GBP 10 nM GBP 10 nM GBP/U0126
0n
M GBP 10 nM GBP 10 nM GBP/U0126
A
B
Fig. 1. Effect of GBP on ERK activation and expression of the TH
and DDC genes in the dorsal integument of day 1 last instar larvae
of the armyworm. (A) Western blots of ERK. The antibody
employed specifically recognizes dually phosphorylated (activated)
ERKs. Phosphorylation of ERK was observed in the integument
after incubation with 10 n
M GBP at 25 °C for 5 min. A MEK inhibi-
tor (10 l
M, U0126) inhibited GBP-induced phosphorylation of ERK.
Each bar indicates the mean ± SD of three independent determina-
tions. *Significantly different from control (0 n
M GBP; P < 0.05,
Student’s t-test). (B) RT-PCR analysis of TH and DDC gene expres-
sion. Clear bands of TH and DDC were expressed in the integu-
ment after incubation with 10 n
M GBP, and expression of both
enzyme genes was not inhibited by U0126. dpERK, dually phos-
phorylated ERK. *Significantly different from control (0 n
M GBP;
P < 0.05, Student’s t-test).

Y. Ninomiya et al. Growth-blocking peptide signaling pathways
FEBS Journal 275 (2008) 894–902 ª 2008 The Authors Journal compilation ª 2008 FEBS 895
insect cells instead of the epidermal integuments,
because we had observed that addition of chemicals
and long incubation caused unexpected damage to the
isolated integument. To select the best cultured cells
for following studies, we tested both enzyme gene
expression and ERK activation of some cultured cells,
including Sf9, MaBr3, and MaBr4 cells, and found
that only MaBr4 cells show clear expression of both
enzyme genes and the ERK activation, as shown in
Fig. 2. Activation of ERK was observed 8 min after
the cells were simply transferred from 4 to 25 °C
(Fig. 2A), and at that time, the TH and DDC genes
were clearly expressed in the cells (Fig. 2B). Further-
more, the GBP-induced gene expression of both
enzymes was highly enhanced only in the Ca
2+
-con-
taining medium (Fig. 2C).
To check whether GBP elevates cytoplasmic Ca
2+
concentrations in MaBr4 cells, Ca
2+
concentrations in
those cells were monitored using a laser confocal
microscope. Addition of GBP to the incubation med-
ium elevated the cytoplasmic Ca
2+
concentrations in

MaBr4 cells, but BSA did not change the Ca
2+
con-
centrations at all (Fig. 3), indicating that, in analogy
to the epidermal cells, MaBr4 cells have the property
of increasing cytoplasmic Ca
2+
concentrations by
GBP [10].
GBP showed a dose-dependent capacity to activate
ERK, as shown in Fig. 4A. The activation of ERK
was roughly proportional to the concentration of GBP
up to 100 nm. Furthermore, GBP-induced activation
of ERK was detected with the presence of EGTA
(Fig. 4B). Whereas a slight enhancement of ERK
phosphorylation was also observed when the concen-
tration of Ca
2+
added to the medium exceeded that of
EGTA (Fig. 4B), the calcium ionophore A23187 did
not activate ERK at all (Fig. 4C). These results imply
that GBP stimulates more than one signaling pathway
leading to activation of ERK, and that among them,
at least one pathway is Ca
2+
-independent.
GBP-induced ERK activation requires MEK
The GBP-induced activation of ERK was observed
transiently 5 min after addition of GBP to the culture
medium of MaBr4 cells (Fig. 5A). This GBP-depen-

dent ERK activation was completely blocked by 10 lm
U0126 (MEK inhibitor), indicating that the GBP sig-
naling pathway requires MEK activity (Fig. 5B).
Furthermore, to test whether the GBP-induced
expression of TH and DDC genes requires ERK acti-
vation, expression of both enzyme genes was measured
in MaBr4 cells stimulated by GBP in the presence of
U0126. GBP clearly enhanced expression of both
enzyme genes even in the presence of 10 lm U0126,
indicating that the GBP signaling pathway for the
stimulation of TH and DDC gene expression does not
require the activated ERK (Fig. 5C).
Analysis of GBP signaling pathways
It is now apparent that the GBP signaling pathways
diverge from a certain component into at least two
pathways: one towards ERK activation, and the
other towards TH and DDC gene expression. To
On ice for 1h/25
o
C incubation time
dpERK
ERK
Marker TH DDC Actin
TH
DDC
Actin
6 min 8 min
**
*
10 nM GBP

10
nM GBP 10 nM BSA
3
mM CaCl
2
3 mM CaCl
2
3 mM CaCl
2
2 mM EGTA 2 mM EGTA 2 mM EGTA 2 mM EGTA 2 mM EGTA
5
4
3
2
1
0
Relative expression
A
B
C
Fig. 2. ERK activation and expression of TH and DDC genes in
MaBr4 cells. (A) Western blots of ERK. Phosphorylation of ERK in
Mabr4 cells was observed 8 min after transfer from 4 to 25 °C. (B)
RT-PCR analysis of expression of TH and DDC genes. RNAs were
prepared from MaBr4 cells 8 min after transfer from 4 to 25 °C. (C)
RT-PCR analysis of GBP-induced expression of TH and DDC genes.
Total RNA was prepared from MaBr4 cells after incubation with
the indicated chemicals at 25 °C for 6 h. Each bar indicates the
mean ± SD of three independent determinations. *Significantly
different from control (0 n

M GBP; P < 0.05, Student’s t-test).
**Significantly different from control (0 n
M GBP; P < 0.01,
Student’s t-test).
Growth-blocking peptide signaling pathways Y. Ninomiya et al.
896 FEBS Journal 275 (2008) 894–902 ª 2008 The Authors Journal compilation ª 2008 FEBS
characterize these pathways, the possible participation
of some components of a classic signal transduction
cascade was tested. Neither elevation of the gene
expression of both enzymes nor ERK activation was
observed after the addition of 2 lm U73122, a phos-
pholipase C (PLC) inhibitor, to the MaBr4 cell culture
medium (Fig. 6). However, whereas the addition of
10 lm chelerythrine chloride, a protein kinase C
(PKC) inhibitor, did not abolish the GBP-dependent
expression of both enzyme genes, it did block the
ERK activation (Fig. 6). In contrast, the addition of
10 lm TMB-8, an inositol triphosphate (IP
3
) receptor
antagonist, did not inhibit the GBP-induced ERK acti-
vation, but abolished the elevation of expression of
both enzyme genes (Fig. 6).
These results clearly indicate that the GBP signaling
events initially activated PLC, which activates ERK
through activation of PKC. Furthermore, IP
3
produced
by activated PLC initiates the signaling cascade that
ultimately triggers Ca

2+
entry from the extracellular
fluid. The elevation of the cytoplasmic Ca
2+
concen-
tration enhances the expression of TH and DDC genes
(Fig. 7).
Discussion
In the present study, we demonstrated that GBP
induces dual phosphorylation (activation) of ERK in
the integuments of armyworm larvae. This observa-
tion, together with a previous report indicating that
robust induction of DDC gene expression requires
activated ERK–MAP kinase in wounded Drosophila
embryos [17], suggested the possibility that the GBP-
induced enhancement of expression of TH and DDC
genes also requires activated ERK. To assess this pos-
sibility, the GBP–ERK pathway was examined in epi-
dermal integuments. Addition of the MEK inhibitor
U0126 to the isolated integument culture medium com-
pletely blocked GBP-induced ERK phosphorylation in
the tissues, but expression of the TH and DDC genes
was enhanced. These results were interpreted to
mean that the GBP signaling pathway towards the
A
(a) (b) (c)
B
(a) (b) (c)
Fig. 3. Monitoring cytoplasmic Ca
2+

concentrations in MaBr4 cells incubated with BSA (A) and GBP (B). Ca
2+
concentrations in MaBr4 cells
containing Fluo-3AM were monitored at 5 min after addition of 10 n
M BSA or GBP. (a) Laser transmission images. (b) Ca
2+
indicator Fluo-
3AM fluorescence images (green). (c) Fluorescence images overlaid with the laser transmission images.
Y. Ninomiya et al. Growth-blocking peptide signaling pathways
FEBS Journal 275 (2008) 894–902 ª 2008 The Authors Journal compilation ª 2008 FEBS 897
expression of both enzyme genes is mediated indepen-
dently of ERK activation. To assess this interpretation,
follow-up analysis was conducted using cultured
MaBr4 insect cells.
Although MaBr4 cells were originally derived from
the larval fat body of Mamestra brassicae, a species
closely related to P. separata, their morphology is
rather hemocyte-like [18]. GBP-induced activation of
dpERK

ERK
dpER
K

ERK
Relative ERK activity and Total ERK amount
4


2

0





4


2


0






4

2

0
*
*
*
*

*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
dpERK

ERK
0 nM GBP 1 nM GBP 10 nM GBP 100 nM GBP
2
mM
EGTA 2
mM
EGTA
10

nM
GBP
2
mM
EGTA/3
mM
CaCl
2
10
nM
GBP
10
nM
GBP
0 0.5 5
DMSO
A23187 concentration (µM)
A
B
C
Fig. 4. Effect of Ca
2+
on GBP-induced activation of ERK in MaBr4
cells. (A) Effects of various concentrations of GBP on ERK phos-
phorylation. MaBr4 cells were incubated with GBP at 25 °C for
5 min. *Significantly different from control (0 n
M GBP; P < 0.05,
Student’s t-test). (B) Requirement of Ca
2+
for GBP-induced phos-

phorylation of ERK. MaBr4 cells were incubated with the indicated
chemicals at 25 °C for 5 min. **Significantly different from control
(0 n
M GBP; P < 0.01, Student’s t-test). (C) Effect of various concen-
trations of the calcium ionophore A23187 on ERK phosphorylation.
MaBr4 cells were incubated with various concentrations of A23187
at 25 °C for 5 min. Because A23187 was dissolved in dimethylsulf-
oxide, dimethylsulfoxide (final concentration 0.1%) was added to
the incubation medium. Only the control incubation contained GBP
(10 n
M). **Significantly different from control (10 nM GBP;
P < 0.01, Student’s t-test). Each bar indicates the mean ± SD of
three independent determinations.
dpERK

ERK
dpERK

ERK
Relative expression
5
4
3
2
1
0
Relative ERK activity

4
0

2
4
2
0
4
2
0
4
0
2
Total ERK
Relative ERK activity

Total ERK
TH
DDC
Actin
*
*
*
*
**
**
**
**
0 5 30 360 min
GBP –
–
+
+

-
–
+
+
-
–
+
+
0 5 30 360 min
GBP -
–
+
+
-
–
+
+
-
–
+
+
U0126 +
+
+
+
+
+
+
+
+

+
+
+
+
+
EGTA EGTA/CaCl
2
GBP -
–
+
+
-
–
+
+
U0126 +
+
+
+
+
+
+
+
A
B
C
Fig. 5. Effect of MEK inhibitor on GBP-induced ERK activation and
enhancement of TH and DDC gene expression inMaBr4 cells. (A)
Effect of incubation time on GBP-induced phosphorylation of ERK.
MaBr4 cells were incubated with or without 10 n

M GBP in the
medium containing 0.1% dimethylsulfoxide at 25 °C. Phosphoryla-
tion of ERK was observed 5 min after addition of GBP to the
MaBr4 incubation medium. Closed and open bars: with and without
GBP, respectively. **Significantly different from control (0 min;
P < 0.01, Student’s t-test). (B) Effect of MEK inhibitor on
GBP-induced ERK phosphorylation. U0126 (10 l
M), a MEK inhibitor,
completely inhibited GBP-induced ERK phosphorylation. Other
explanations as in (A). (C) Effect of MEK inhibitor on enhancement
of TH and DDC gene expression. MaBr4 cells were incubated at
25 °C for 6 h after addition of the indicated chemicals, and total
RNA was prepared. The GBP-induced expression of TH and DDC
mRNAs was not blocked by the MEK inhibitor. *Significantly differ-
ent from control (0 n
M GBP; P < 0.05, Student’s t-test). Each bar
indicates the mean ± SD of three independent determinations.
Growth-blocking peptide signaling pathways Y. Ninomiya et al.
898 FEBS Journal 275 (2008) 894–902 ª 2008 The Authors Journal compilation ª 2008 FEBS
ERK was transiently observed at 5 min after addition
of GBP to the incubation medium of MaBr4 cells. The
involvement of a MAP kinase kinase (MEK) in the
ERK activation pathway [19] was tested by evaluating
the effect of U0126. U0126 completely abolished the
temperature-dependent activation of ERK, indicating
that ERK activation in MaBr4 cells proceeds through
the common pathway characterized in mammalian
cells.
Prior studies indicated that GBP induces Ca
2+

influx into brain synaptosomes and epidermal cells
[10,20]. Especially in the latter, the enhancement of
TH and DDC gene expression was demonstrated to be
accompanied by GBP-induced elevation of the cyto-
plasmic Ca
2+
concentration. We examined whether the
GBP-induced activation of ERK requires Ca
2+
influx
into MaBr4 cells. Excess amounts of EGTA did not
block the GBP-induced ERK activation. Furthermore,
the calcium ionophore A23187 did not activate ERK
at all, even at a concentration of 5 lm, suggesting that
the GBP-dependent activation of ERK does not
require Ca
2+
influx into MaBr4 cells. However, the
GBP-induced enhancement of TH and DDC gene
expression was completely abolished by EGTA. Fur-
thermore, the GBP-dependent expression of both
enzyme genes was observed in the presence of the
MEK inhibitor, thereby demonstrating that the GBP-
dependent enhancement of expression of the enzyme
genes in MaBr4 cells is independent of the ERK–MAP
kinase pathway.
As U73122 (PLC inhibitor) has been shown to block
the formation of IP
3
in several insect tissues [21,22],

we applied it to MaBr4 cells. The inhibitory effect of
U72122 on ERK and expression of the enzyme genes
clearly demonstrated that GBP signaling events ini-
tially activate PLC. The PLC-triggered metabolism of
membrane phospholipids produces diacylglycerol
(DAG) and IP
3
. Because chelerythrine chloride, a
PKC inhibitor, abolished GBP-dependent activation of
ERK, the PLC-produced DAG would contribute to
activation of PKC [23–25]. In contrast, the observation
that TMB-8, an IP
3
receptor antagonist, blocked GBP-
TH
DDC
Actin
Control PLC inhibitor IP
3R antagonist PKC inhibitor
*
*
*
*
Relative gene expression of
TH , DDC , and actin
5
4
3
2
1

0
Control PLC inhibitor IP3R antagonist PKC inhibitor
*
*
*
*
dpERK
ERK
Relative ERK activity and
total ERK amount
5
4
3
2
1
0
GBP +
+ –– + – + – +
GBP +
+ –– + – + – +
Fig. 6. ERK activation and TH and DDC gene expression in the
presence of various inhibitors. MaBr4 cells were incubated with or
without 10 n
M GBP in the medium containing 0.1% dimethylsulfox-
ide with 2 l
M U73122 (PLC inhibitor), 10 lM TMB-8 (IP
3
receptor
antagonist), or 10 l
M chelerythrine chloride (PKC inhibitor) at 25 °C

for 6 h, and total RNA was prepared from cells after incubation.
Phosphorylation of ERK was observed 5 min after addition of
10 n
M GBP to the MaBr4 incubation medium. Each bar indicates
the mean ± SD of three independent determinations. *Significantly
different from control (0 n
M GBP; P < 0.05, Student’s t-test). **Sig-
nificantly different from control (0 n
M GBP; P < 0.01, Student’s
t-test).
Fig. 7. Schematic representation of hypothesized GBP signaling
pathways in MaBr4 cells. Stimulation of the putative GBP receptor
activates PLC. DAG, newly formed by activated PLC, stimulates
PKC, and activated PKC induces ERK activation. Independently of
this signaling pathway, the IP
3
produced by activated PLC stimu-
lates the IP
3
receptor to release Ca
2+
from the endoplasmic retic-
ulum, which leads to the extracellular Ca
2+
influx that induces
robust enhancement of TH and DDC gene expression. ER, endo-
plasmic reticulum; GBPR, growth-blocking peptide receptor; IP
3
R,
IP

3
receptor.
Y. Ninomiya et al. Growth-blocking peptide signaling pathways
FEBS Journal 275 (2008) 894–902 ª 2008 The Authors Journal compilation ª 2008 FEBS 899
dependent expression of both enzyme genes suggested
that the IP
3
receptor-mediated Ca
2+
influx from the
extracellular fluid plays an essential role in the tran-
scriptional enhancement of TH and DDC genes
(Fig. 7). Although the GBP signaling pathways were
characterized by analyzing MaBr4 cells, it is reason-
able to assume that these pathways are employed in
armyworm epidermal cells. It has been reported that
prothoracicotropic hormone (PTTH) stimulates Ca
2+
influx into the prothoracic gland and also activates
ERK in the tobacco hornworm Manduca sexta
[26–28]. PTTH-induced ERK activation is strongly
dependent on external Ca
2+
, and ERK activation is
induced by substitution of the calcium ionophore
A23187 for PTTH. Therefore, the PTTH-induced
events in the prothoracic gland seem to be completely
different from those observed in the GBP signaling
pathway in the epidermis and MaBr4 cells.
Once GBP was identified as a growth inhibitory pep-

tide in insects, a series of follow-up studies revealed its
diverse biological functions, including paralysis induc-
tion, cell proliferation, and stimulation of immune cells
[29–31]. This study clearly characterized two different
signaling pathways of GBP in epidermal cells and cul-
tured MaBr4 cells. Although the multiple activities
reported for GBP may reflect differences in the types
of GBP receptors expressed by specific tissues or devel-
opmental stages of insects, divergence of the GBP
signaling pathway must also enable GBP to exert
different activities in the same cell.
In summary, this study provides unequivocal evi-
dence that GBP stimulates Ca
2+
influx into cultured
MaBr4 cells and subsequent enhancement of expres-
sion of the TH and DDC genes. Furthermore, GBP
stimulates ERK phosphorylation, which is not essen-
tial for the enhancement of expression of either
enzyme gene. One of the GBP pathway components
is PLC, from which the downstream signaling path-
ways diverge to ERK activation through PKC and
enzyme gene expression through IP
3
receptor-medi-
ated Ca
2+
influx. Therefore, it is reasonable to con-
clude that the GBP signaling pathway to expression
of the TH and DDC genes is unique in its ability to

function independently of the ERK–MAP kinase
kinase pathway.
Experimental procedures
Animals
Pseudaletia separata larvae were reared on an artificial diet
at 25 ± 1 °C with a 16 h light ⁄ 8 h dark photoperiod [10].
Penultimate instar larvae undergoing ecdysis between 4 and
4.5 h after starting the light period were designated as
day 0 last instar larvae.
Chemicals
Polyclonal antibody to MAP kinase (ERK-1, ERK-2),
U0126, U73122, TMB-8 and chelerythrine chloride were
purchased from Sigma-Aldrich Co. (St Louis, MO, USA).
Monoclonal antibody to active MAP kinase was obtained
from Promega Co. (Madison, WI, USA). A23187 (calcium
ionophore) and Grace’s medium (Cat. No. 11595-030) were
purchased from Nacalai Tesque Co. (Kyoto, Japan) and
Invitrogen Co. (Carlsbad, CA, USA), respectively. All other
chemicals were of reagent grade.
Dissection and culture of integument
A whole abdominal integument between the first and sec-
ond segments was dissected from the test day 1 last instar
larva of the armyworm. Care was taken to remove all the
adhering fat body tissue from the integument. The dissected
integument was separated into dorsal and ventral parts.
After being washed with NaCl ⁄ P
i
, the tissues were lightly
blotted with filter paper, weighed, and immediately used for
experiments.

Pieces of dorsal larval integument were cultured in
Grace’s medium with or without 10 nm GBP at 25 °C. As
a control, 10 nm BSA was added to the medium. To
remove extracellular free Ca
2+
, Grace’s medium containing
1mm EGTA was used. A23187 was dissolved in dimethyl-
sulfoxide and added to the medium.
Cell culture
The MaBr4 cell line established from M. brassicae fat
bodies was kindly provided by T. Hiraoka (Tokyo Univer-
sity of Agriculture and Technology), and maintained in
Mitsuhashi ⁄ Maramorosh insect medium (MM medium) at
25 °C [18].
RT-PCR and quantitative PCR
Total RNA was isolated from integuments of test tissues or
cultured cells using TRIzol reagent (Gibco-BRL, Rockville,
MD, USA), according to the manufacturer’s instructions.
Two micrograms of total RNA was reverse transcribed
with oligo(dT) primer using ReverTra Ace (Toyobo, Osaka,
Japan). The cDNA was amplified with a TH-specific primer
pair (5¢-CAGCTGCCCAGAAGAACCGCGAGATG-3¢,
+11 to +36 bp and 5¢-GAACTCCACGGTGAACCAGT-
3¢, +1286 to +1305 bp), a DDC-specific primer pair
(5¢-ATGGAGGCCGGAGATTTCAAAG-3¢, +1 to +22
bp and 5¢-ACGGGCTTTAAGTATTTCATCAGGC-3¢,
+1405 to +1428 bp), and an actin primer pair (5¢-TTCG
Growth-blocking peptide signaling pathways Y. Ninomiya et al.
900 FEBS Journal 275 (2008) 894–902 ª 2008 The Authors Journal compilation ª 2008 FEBS
AGCAGGAGATGGCCACC-3¢ and 5¢-GAGATCCACAT

CTGYTGGAAGGT-3¢). PCR was conducted under the
following conditions: 25 cycles at 94 °C for 1 min, 50 °C
for 1 min, and 72 °C for 2 min.
Real-time quantitative PCR was used to determine the
relative expression levels of TH and DDC. Quantitative
PCR was carried out with 2.5% of the reverse transcription
product in a 20 lL reaction volume of LightCycler Fast
DNA Master SYBR Green I (Roche Applied Science, Indi-
anapolis, IN, USA), using the Light-Cycler 1.2 instrument
and software (Roche Applied Science).
Immunoblotting analysis
Integuments dissected from larvae or cultured cells were
homogenized in 80 mm Tris ⁄ HCl buffer (pH 8.8) contain-
ing 1% SDS and 2.5% 2-mercaptoethanol, and centrifuged
at 20 000 g for 10 min at 4 °C. The supernatant was boiled
for 5 min and applied to SDS ⁄ PAGE gel. Proteins sepa-
rated by SDS ⁄ PAGE were electrically transferred to a
poly(vinylidene difluoride) membrane filter, blocked, and
probed with the indicated primary antibody. After being
washed thoroughly with 0.05% Tween-20 in Tris-buffered
saline (10 mm, 150 mm NaCl, pH 7.5), antigens were
detected using peroxidase-conjugated secondary antibody
and a 4-chloro-1-naphtol (4CN) Immun-Blot Colorimetric
Assay kit (Bio-Rad Laboratories, Hercules, CA, USA) [32].
All positive bands were quantified using imagej (NIH).
Confocal calcium imaging
MaBr4 cells were washed with Ca
2+
-free Carlson solution
(120 mm NaCl, 2.7 mm KCl, 0.5 mm MgCl

2
, 1.7 mm
NaH
2
PO
4
, 1.4 mm NaHCO
3
, 2.2 mm glucose), and loaded
with 10 lm Fluo-3AM (Dojindo Laboratories, Kumamoto,
Japan) at 25 °C for 30 min. After loading, the cells were
washed twice in Ca
2+
-free Carlson solution by sedimenta-
tion and resuspension, and placed on the slide glass. The
cells were stimulated with Grace’s medium with or without
1nm GBP and immediately excited with light of 488 nm
wavelength by a confocal imaging system CellMap (Carl
Zeiss, Oberkochen, Germany).
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