Ecdysteroidogenesis In Prothoracic Glands Research Articles

Signaling in cAMP-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori

In the present study, we investigated downstream pathways of cyclic adenosine monophosphate (cAMP) signaling (which is related to prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis) in Bombyx mori prothoracic glands (PGs). Results showed that treatment with either dibutyryl cAMP (dbcAMP) or 1-methyl-3-isobutylxanthine (MIX) inhibited phosphorylation of adenosine 5′-monophosphate-activated protein kinase (AMPK) and activated phosphorylation of the translational repressor, 4E-binding protein (4E-BP), a marker of target of rapamycin (TOR) signaling. A chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, AICAR) increased dbcAMP-inhibited AMPK phosphorylation and blocked dbcAMP-stimulated phosphorylation of 4E-BP, indicating that inhibition of AMPK phosphorylation lies upstream of dbcAMP-stimulated TOR signaling. Treatment of PGs with dbcAMP and MIX also stimulated phosphorylation of a 37-kDa protein, as recognized by a protein kinase C (PKC) substrate antibody, indicating that cAMP activates PKC signaling. Treatment with either LY294002 or AICAR did not affect dbcAMP-stimulated phosphorylation of the PKC-dependent 37-kDa protein, indicating that cAMP-stimulated PKC signaling is not related to phosphoinositide 3-kinase (PI3K) or AMPK. In addition, dbcAMP-stimulated ecdysteroidogenesis in PGs was partially inhibited by pretreatment with either LY294002, AICAR, or calphostin C. From these results, we concluded that AMPK/TOR/4E-BP and PKC pathways are involved in ecdysteroidogenesis of PGs stimulated by cAMP signaling in B. mori.

Expression of tyrosine phosphatases in relation to PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori

Protein tyrosine phosphorylation is a reversible, dynamic process regulated by the activities of tyrosine kinases and tyrosine phosphatases. Although the involvement of tyrosine kinases in the prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs) has been documented, few studies have been conducted on the involvement of protein tyrosine phosphatases (PTPs) in PTTH-stimulated ecdysteroidogenesis. In the present study, we investigated the correlation between PTPs and PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs. Our results showed that the basal PTP enzymatic activities exhibited development-specific changes during the last larval instar and pupation stage, with high activities being detected during the later stages of the last larval instar. PTP enzymatic activity was stimulated by PTTH treatment both in vitro and in vivo. Pretreatment with phenylarsine oxide (PAO) and benzylphosphonic acid (BPA), two chemical inhibitors of tyrosine phosphatase, reduced PTTH-stimulated enzymatic activity. Determination of ecdysteroid secretion showed that treatment with PAO and BPA did not affect basal ecdysteroid secretion, but greatly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated PTP activity is indeed involved in ecdysteroid secretion. PTTH-stimulated phosphorylation of the extracellular signal-regulated kinase (ERK) and 4E-binding protein (4E-BP) was partially inhibited by pretreatment with either PAO or BPA, indicating the potential link between PTPs and phosphorylation of ERK and 4E-BP. In addition, we also found that in vitro treatment with 20-hydroxyecdysone did not affect PTP enzymatic activity. We further investigated the expressions of two important PTPs (PTP 1B (PTP1B) and the phosphatase and tension homologue (PTEN)) in Bombyx PGs. Our immunoblotting analysis showed that B. mori PGs contained the proteins of PTP1B and PTEN, with PTP1B protein undergoing development-specific changes. Protein levels of PTP1B and PTEN were not affected by PTTH treatment. The gene expression levels of PTP1B and PTEN showed development-specific changes. From these results, we suggest that PTTH-regulated PTP signaling may crosstalk with ERK and target of rapamycin (TOR) signaling pathways and is a necessary component for stimulation of ecdysteroid secretion.

Reactive oxygen species-mediated bombyxin signaling in Bombyx mori

In the present study, we demonstrated that bombyxin, an insect insulin-like peptide, modulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs) through redox signaling. Our results showed that bombyxin treatment resulted in a transient increase in intracellular reactive oxygen species (ROS) concentration, as measured using 2′,7′-dichlorofluorescin diacetate (DCFDA), an oxidation-sensitive fluorescent probe. The antioxidant N-acetylcysteine (NAC) abolished the bombyxin-induced increase in fluorescence in Bombyx PGs. Furthermore, bombyxin-induced ROS production was inhibited by mitochondrial oxidative phosphorylation inhibitors (rotenone and antimycin A), indicating mitochondria-mediated ROS production. The stimulation of ROS production in response to bombyxin appears to undergo development-specific changes. We further investigated the action mechanism of bombyxin-stimulated ROS signaling. Results showed that in the presence of either NAC, rotenone, or antimycin A, bombyxin-stimulated phosphorylation of insulin receptor, Akt, and 4E-binding protein (4E-BP) was blocked and bombyxin-stimulated ecdysteroidogenesis in PGs was greatly inhibited. From these results, we conclude that ROS signaling appears to be involved in bombyxin-stimulated ecdysteroidogenesis of PGs in B. mori by modulating the phosphorylation of insulin receptor, Akt, and 4E-BP. To our knowledge, this is the first demonstration of redox regulation in insulin signaling in an insect system.

Involvement of reactive oxygen species in PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori

In the present study, the possible involvement of reactive oxygen species (ROS) in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis of Bombyx mori prothoracic glands (PGs) was investigated. Results showed that PTTH treatment resulted in a rapidly transient increase in the intracellular ROS concentration, as measured using 2′,7′-dichlorofluorescin diacetate (DCFDA), an oxidation-sensitive fluorescent probe. The antioxidant, N-acetylcysteine (NAC), abolished PTTH-induced increase in fluorescence. Furthermore, PTTH-induced ROS production was partially inhibited by the NAD(P)H oxidase inhibitor, apocynin, indicating that NAD(P)H oxidase is one of the sources for PTTH-stimulated ROS production. Four mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and diphenylene iodonium (DPI)) significantly attenuated ROS production induced by PTTH. These data suggest that the activity of complexes I and III in the electron transport chain and the mitochondrial inner membrane potential (ΔΨ) contribute to PTTH-stimulated ROS production. In addition, PTTH-stimulated ecdysteroidogenesis was greatly inhibited by treatment with either NAC or mitochondrial inhibitors (rotenone, antimycin A, FCCP, and DPI), but not with apocynin. These results indicate that mitochondria-derived, but not membrane NAD(P)H oxidase-mediated ROS signaling, is involved in PTTH-stimulated ecdysteroidogenesis of PGs in B. mori.

DECREASED JH BIOSYNTHESIS IS RELATED TO PRECOCIOUS METAMORPHOSIS IN RECESSIVE TRIMOLTER (rt) MUTANTS OF THE SILKWORM, Bombyx mori

In recessive trimolter (rt) mutants of the silkworm, Bombyx mori, that have four larval instars rather than five larval instars of normal B. mori, a decrease after a small increase in the hemolymph ecdysteroid titer during the early stages of the last (fourth) larval instar appeared to be a prerequisite for larvae to undergo precocious metamorphosis. The present study was carried out to investigate the possible mechanism underlying this decrease in the ecdysteroid titer. It was found that juvenile hormone (JH) biosynthetic activity of the corpora allata (CA) increased during the first day of the last larval instar, but its absolute JH biosynthesis activity was relatively lower compared to that of normal fourth-instar larvae in tetramolters. This lowered JH biosynthetic activity appeared to be related to a decrease in prothoracic gland ecdysteroidogenesis during the second day of the last instar, because hydroprene application prevented this decrease in prothoracic gland ecdysteroidogenesis, leading to the induction of a supernumerary larval molt. The in vitro incubation of prothoracic glands with hydroprene showed that hydroprene did not directly exert its action on prothoracicotropic hormone (PTTH) release. Further study showed that the application of hydroprene enhanced the competency of the glands to respond to PTTH. From these results, it was supposed that the lowered JH biosynthesis of the CA during the first day of last instar in rt mutants was related to decreased ecdysteroidogenesis in the prothoracic glands during the second day, thus playing a role in leading to precocious metamorphosis.

TOR signaling is involved in PTTH-stimulated ecdysteroidogenesis by prothoracic glands in the silkworm, Bombyx mori

The prothoracicotropic hormone (PTTH) is a stimulator of ecdysteroidogenesis in prothoracic gland of larval insects. Our recent studies showed that phosphoinositide 3-kinase (PI3K)/Akt signaling was involved in PTTH-stimulated ecdysteroidogenesis by Bombyx mori prothoracic glands. In the present study, downstream signaling of PI3K/Akt was further investigated. Results showed that PTTH rapidly enhanced the phosphorylation of translational repressor 4E-binding protein (4E-BP) and p70 ribosomal protein S6 kinase (S6K), two known downstream signaling targets of the target of rapamycin complex 1 (TORC1). PTTH stimulated 4E-BP phosphorylation in time- and dose-dependent manners. Injection of PTTH into day-6 last instar larvae greatly increased 4E-BP phosphorylation, verifying the in vitro effect. PTTH-stimulated 4E-BP phosphorylation was blocked by both LY294002 and wortmannin, indicating the involvement of PI3K. Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors (PD 98059 and U0126), did not inhibit PTTH-stimulated 4E-BP phosphorylation, implying that ERK signaling is not related to PTTH-stimulated 4E-BP phosphorylation. The phosphorylation of S6K was also stimulated by PTTH both in vitro and in vivo. PI3K signaling appears to be involved in PTTH-stimulated phosphorylation of S6K. Rapamycin, a specific inhibitor of mammalian TOR signaling attenuated PTTH-stimulated phosphorylation of 4E-BP and S6K of the glands, and greatly inhibited PTTH-stimulated ecdysteroidogenesis. Examination of gene expression levels of 4E-BP and S6K showed that PTTH inhibited mRNA levels of both 4E-BP and S6K, indicating that PTTH may exert its action at both the transcriptional and phosphorylation levels. These results suggest that PTTH/PI3K/TOR/4E-BP (S6K) signaling is involved in PTTH-stimulated ecdysteroidogenesis by prothoracic glands in B. mori.

Autocrine activation of ecdysteroidogenesis in the prothoracic glands of the silkworm, Bombyx mori

Ecdysteroidogenesis in the prothoracic glands is activated by the neuropeptide, prothoracicotropic hormone (PTTH). The present study demonstrates autocrine activation of ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori. Using both a long-term in vitro organ culture system and an ecdysteroid radioimmunoassay, it was found that either decreasing the incubation volume, from 100 to 5 μl, or increasing the number of glands incubated per drop (50 μl) from 1 to 5 significantly increased ecdysteroid secretion. Prothoracic gland-conditioned medium was used to clarify the autocrine factor. The results showed that activation of ecdysteroidogenesis by the prothoracic gland-conditioned medium appeared to be dose dependent and a dramatic increase in ecdysteroid secretion was observed after 6 h of incubation in the conditioned medium. Moreover, it appeared that autocrine activation occurred when glands were incubated in large volumes of incubation medium and during a short incubation period, indicating that the factor may exert its action in situ at some specific developmental stages. This tropic factor was further characterized, and it was found that the factor seemed to be heat-stable, with a molecular weight estimated to be between 1000 and 3000 Da. Injection of the concentrated putative autocrine factor into day 5 last instar larvae greatly increased ecdysteroidogenic activity of the prothoracic glands compared to those injected with saline, indicating the possible in vivo function of the present factor.

The ecdysteroidogenic P450 Cyp302a1/disembodied from the silkworm, Bombyx mori, is transcriptionally regulated by prothoracicotropic hormone

During larval and pupal development of insects, ecdysone is synthesized in the prothoracic gland (PG). Although several Drosophila genes, including Halloween P450 genes, are known to be important for ecdysteroidogenesis in PG, little is known of the ecdysteroidogenic genes in other insects. Here we report on Cyp302a1/disembodied (dib-Bm), one of the Halloween P450s in the silkworm Bombyx mori that is a carbon-22 hydroxylase. dib-Bm is predominantly expressed in PG and its developmental expression profile is correlated with a change in the ecdysteroid titre in the haemolymph. Furthermore, dib-Bm expression in cultured PGs is significantly induced by treatment with prothoracicotropic hormone. This is the first report on the transcriptional induction of a steroidogenic gene by the tropic hormone in insects.

The diapause hormone-pheromone biosynthesis activating neuropeptide gene of Helicoverpaarmigera encodes multiple peptides that break, rather than induce, diapause

FXPRLamide peptides encoded by the DH-PBAN (diapause hormone-pheromone biosynthesis activating neuropeptide) gene induce embryonic diapause in Bombyx mori, but terminate pupal diapause in Helicoverpa armigera (Har). Here, we explore the mechanisms of terminating pupal diapause by the FXPRLamide peptides. Using quantitative RT-PCR, we observed that expression of Har-DH-PBAN mRNA in the SG of nondiapause-type pupae was significantly higher than in diapause-type pupae. Immunocytochemical results indicated that the level of FXPRLamide peptides and axonal release are related to the diapause decision. Ecdysteroidogenesis in prothoracic glands (PGs) was stimulated by synthetic Har-DH in vivo and in vitro, and labeled Har-DH bound to the membrane of the PG, thus suggesting that DH breaks diapause by activating the PG to synthesize ecdysone. Furthermore, the response of DH in terminating diapause was temperature dependent. Decerebration experiments showed that the brain can control pupal development through the regulation of DH, and DH can terminate diapause and promote development without the brain. This result suggests a possible mechanism of response for the signals of DH and other FXPRLamide peptides in H. armigera.