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: hayakayo@cc.saga-u.ac.jp (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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