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Pigment dispersing factor regulates ecdysone biosynthesis via bombyx neuropeptide G protein coupled receptor-B2 in the prothoracic glands of Bombyx mori.

Iga M, Nakaoka T, Suzuki Y, Kataoka H - PLoS ONE (2014)

Bottom Line: Furthermore, we identified PDF as a ligand for BNGR-B2.PDF did not affect the transcript level of known ecdysone biosynthetic enzymes, and inhibiting transcription did not suppress ecdysone biosynthesis, suggesting that the effects of PDF might be mediated by translational regulation and/or post-translational modification.In addition, the participation of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), target of rapamycin (TOR) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) in the PDF signaling pathway was discovered.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan.

ABSTRACT
Ecdysone is the key hormone regulating insect growth and development. Ecdysone synthesis occurs in the prothoracic glands (PGs) and is regulated by several neuropeptides. Four prothoracicotropic and three prothoracicostatic factors have been identified to date, suggesting that ecdysone biosynthesis is intricately regulated. Here, we demonstrate that the neuropeptide pigment dispersing factor (PDF) stimulates ecdysone biosynthesis and that this novel signaling pathway partially overlaps with the prothoracicotropic hormone (PTTH) signaling pathway. We performed transcriptome analysis and focused on receptors predominantly expressed in the PGs. From this screen, we identified a candidate orphan G protein coupled receptor (GPCR), Bombyx neuropeptide GPCR-B2 (BNGR-B2). BNGR-B2 was predominantly expressed in ecdysteroidogenic tissues, and the expression pattern in the PGs corresponded to the ecdysteroid titer in the hemolymph. Furthermore, we identified PDF as a ligand for BNGR-B2. PDF stimulated ecdysone biosynthesis in the PGs, but the stimulation was only observed in the PGs during a specific larval stage. PDF did not affect the transcript level of known ecdysone biosynthetic enzymes, and inhibiting transcription did not suppress ecdysone biosynthesis, suggesting that the effects of PDF might be mediated by translational regulation and/or post-translational modification. In addition, the participation of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), target of rapamycin (TOR) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) in the PDF signaling pathway was discovered.

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Screening of candidate receptors.(A) Tissue distribution of BNGR-B2. The expression of BNGR-B2 was measured by RT-PCR in the selected tissues from gut-purged fifth instar larvae (p50T strain). PG: prothoracic gland, BR: brain, FB: fat body, MT: Malpighian tubule, ASG: anterior silk gland, MG: midgut, TE: testis and OV: ovary. (B) Developmental profile of BNGR-B2 in the PGs. The expression of BNGR-B2 was measured by Q-PCR. The timing of molting, gut purge and pupation in our rearing conditions is indicated with arrows. Each datum point represents the mean ±SEM (n = 3). The dashed line indicates the outline of the hemolymph ecdysteroid titer described by Koyama et al., 2004 (4th instar), Sakurai et al., 1998 (5th instar) and Kaneko et al., 2006 (5th instar). (A, B) RpL3 was used as an internal standard.
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pone-0103239-g001: Screening of candidate receptors.(A) Tissue distribution of BNGR-B2. The expression of BNGR-B2 was measured by RT-PCR in the selected tissues from gut-purged fifth instar larvae (p50T strain). PG: prothoracic gland, BR: brain, FB: fat body, MT: Malpighian tubule, ASG: anterior silk gland, MG: midgut, TE: testis and OV: ovary. (B) Developmental profile of BNGR-B2 in the PGs. The expression of BNGR-B2 was measured by Q-PCR. The timing of molting, gut purge and pupation in our rearing conditions is indicated with arrows. Each datum point represents the mean ±SEM (n = 3). The dashed line indicates the outline of the hemolymph ecdysteroid titer described by Koyama et al., 2004 (4th instar), Sakurai et al., 1998 (5th instar) and Kaneko et al., 2006 (5th instar). (A, B) RpL3 was used as an internal standard.

Mentions: The tissue distribution of BNGR-B2 was investigated in the gut-purged fifth instar larvae using RT-PCR. As shown in Figure 1A, BNGR-B2 was predominantly expressed in the PGs and gonads (testis and ovary). The expression profile of BNGR-B2 in the PGs was determined using Q-PCR in fourth and fifth instar larvae and the first day of pupae (Figure 1B). BNGR-B2 expression peaked on day 3 in fourth instar larvae (IV3). Furthermore, during the larval-pupal metamorphosis, the expression of BNGR-B2 began to increase on day 7 and peaked on day 9 in fifth instar larvae (V7 and V9), after which the expression decreased. Thus, the expression profile of BNGR-B2 in the PGs correlated with the ecdysteroid titer in the hemolymph [22]–[24].


Pigment dispersing factor regulates ecdysone biosynthesis via bombyx neuropeptide G protein coupled receptor-B2 in the prothoracic glands of Bombyx mori.

Iga M, Nakaoka T, Suzuki Y, Kataoka H - PLoS ONE (2014)

Screening of candidate receptors.(A) Tissue distribution of BNGR-B2. The expression of BNGR-B2 was measured by RT-PCR in the selected tissues from gut-purged fifth instar larvae (p50T strain). PG: prothoracic gland, BR: brain, FB: fat body, MT: Malpighian tubule, ASG: anterior silk gland, MG: midgut, TE: testis and OV: ovary. (B) Developmental profile of BNGR-B2 in the PGs. The expression of BNGR-B2 was measured by Q-PCR. The timing of molting, gut purge and pupation in our rearing conditions is indicated with arrows. Each datum point represents the mean ±SEM (n = 3). The dashed line indicates the outline of the hemolymph ecdysteroid titer described by Koyama et al., 2004 (4th instar), Sakurai et al., 1998 (5th instar) and Kaneko et al., 2006 (5th instar). (A, B) RpL3 was used as an internal standard.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4114559&req=5

pone-0103239-g001: Screening of candidate receptors.(A) Tissue distribution of BNGR-B2. The expression of BNGR-B2 was measured by RT-PCR in the selected tissues from gut-purged fifth instar larvae (p50T strain). PG: prothoracic gland, BR: brain, FB: fat body, MT: Malpighian tubule, ASG: anterior silk gland, MG: midgut, TE: testis and OV: ovary. (B) Developmental profile of BNGR-B2 in the PGs. The expression of BNGR-B2 was measured by Q-PCR. The timing of molting, gut purge and pupation in our rearing conditions is indicated with arrows. Each datum point represents the mean ±SEM (n = 3). The dashed line indicates the outline of the hemolymph ecdysteroid titer described by Koyama et al., 2004 (4th instar), Sakurai et al., 1998 (5th instar) and Kaneko et al., 2006 (5th instar). (A, B) RpL3 was used as an internal standard.
Mentions: The tissue distribution of BNGR-B2 was investigated in the gut-purged fifth instar larvae using RT-PCR. As shown in Figure 1A, BNGR-B2 was predominantly expressed in the PGs and gonads (testis and ovary). The expression profile of BNGR-B2 in the PGs was determined using Q-PCR in fourth and fifth instar larvae and the first day of pupae (Figure 1B). BNGR-B2 expression peaked on day 3 in fourth instar larvae (IV3). Furthermore, during the larval-pupal metamorphosis, the expression of BNGR-B2 began to increase on day 7 and peaked on day 9 in fifth instar larvae (V7 and V9), after which the expression decreased. Thus, the expression profile of BNGR-B2 in the PGs correlated with the ecdysteroid titer in the hemolymph [22]–[24].

Bottom Line: Furthermore, we identified PDF as a ligand for BNGR-B2.PDF did not affect the transcript level of known ecdysone biosynthetic enzymes, and inhibiting transcription did not suppress ecdysone biosynthesis, suggesting that the effects of PDF might be mediated by translational regulation and/or post-translational modification.In addition, the participation of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), target of rapamycin (TOR) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) in the PDF signaling pathway was discovered.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan.

ABSTRACT
Ecdysone is the key hormone regulating insect growth and development. Ecdysone synthesis occurs in the prothoracic glands (PGs) and is regulated by several neuropeptides. Four prothoracicotropic and three prothoracicostatic factors have been identified to date, suggesting that ecdysone biosynthesis is intricately regulated. Here, we demonstrate that the neuropeptide pigment dispersing factor (PDF) stimulates ecdysone biosynthesis and that this novel signaling pathway partially overlaps with the prothoracicotropic hormone (PTTH) signaling pathway. We performed transcriptome analysis and focused on receptors predominantly expressed in the PGs. From this screen, we identified a candidate orphan G protein coupled receptor (GPCR), Bombyx neuropeptide GPCR-B2 (BNGR-B2). BNGR-B2 was predominantly expressed in ecdysteroidogenic tissues, and the expression pattern in the PGs corresponded to the ecdysteroid titer in the hemolymph. Furthermore, we identified PDF as a ligand for BNGR-B2. PDF stimulated ecdysone biosynthesis in the PGs, but the stimulation was only observed in the PGs during a specific larval stage. PDF did not affect the transcript level of known ecdysone biosynthetic enzymes, and inhibiting transcription did not suppress ecdysone biosynthesis, suggesting that the effects of PDF might be mediated by translational regulation and/or post-translational modification. In addition, the participation of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), target of rapamycin (TOR) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) in the PDF signaling pathway was discovered.

Show MeSH
Related in: MedlinePlus