Some autophagic and apoptotic features of programmed cell death in the anterior silk glands of the silkworm, Bombyx mori

Programmed cell death has been subdivided into two major groups: apoptosis and autophagic cell death. The anterior silk gland of Bombyx mori degenerates during larval-pupal metamorphosis. Our findings indicate that two types of programmed cell death features are observed during this physiological process. During the prepupal period, pyknosis of the nucleus, cell detachment,and membrane blebbing occur and they are the first signs of programmed cell death in the anterior silk glands. According to previous studies, all of these morphological appearances are common for both cell-death types. Autophagy features are also exhibited during the prepupal period. Levels of one of the lysosomal marker enzymes-acid phosphatase-are high during this period then decrease gradually. Vacuole formation begins to appear first at the basal surface of the cell, then expands to the apical surface just before the larval pupal ecdysis. After larval-pupal ecdysis, DNA fragmentation, which is the obvious biochemical marker of apoptosis, is detected in agarose gel electrophoresis, which also shows that caspase-like enzyme activities occur during the programmed cell death process of the anterior silk glands. Apoptosis and autophagic cell death interact with each other during the degeneration process of the anterior silk gland in Bombyx mori and this interaction occurs at a late phase of cell death. We suggest that apoptotic cell death only is not enough for whole gland degeneration and that more effective degeneration occurs with this cooperation.     

Fenoxycarb modulates ecdysone receptor B1 and programmed cell death of the anterior silk gland of silkworm Bombyx mori

Fenoxycarb, O‐ethyl N‐(2‐(4‐phenoxyphenoxy)‐ethyl) carbamate has been shown to be one of the most potent juvenile hormone analogues against a variety of insect species. In the present study, topical application of fenoxycarb to fifth‐instar larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) was performed immediately after the fourth ecdysis (on day 0), day 3 and day 6 of the instar and then its effects on the anterior silk glands (ASG) and ecdysone receptor B1 (EcR‐B1) protein were investigated during larval pupal development. Fenoxycarb application increased the instar length and prevented metamorphic events, depending on the application time. The ASGs of B. mori undergo programmed cell death during the larval–pupal metamorphosis and an insect steroid, 20‐hydroxyecdysone (20E), triggers this cell death. The exact mechanism by which 20E and juvenile hormone regulates programmed cell death in insect tissues is poorly understood. To gain insights into how juvenile hormone regulates metamorphic events like programmed cell death in the anterior silk glands, we analyzed the progression of programmed cell death with morphological observations and biochemical experiments like acid phosphatase activity and DNA electrophoresis. Then we examined the EcR‐B1 protein levels and their relationships with programmed cell death. Our results indicated that fenoxycarb modulates programmed cell death of the anterior silk glands and EcR‐B1 protein level, depending on the application time. Fenoxycarb may exhibit its effects in at least two different ways: (i) acting on prothoracic gland secretory activity; and/or (ii) regulation of EcR‐B1 expression in the anterior silk glands for programmed cell death process.     

Proteomic analysis of silk gland programmed cell death during metamorphosis of the silkworm Bombyx mori

The silk gland of the silkworm Bombyx mori undergoes programmed cell death (PCD) during pupal metamorphosis. On the basis of their morphological changes and the occurrence of a DNA ladder, the tissue cells were categorized into three groups: intact, committed, and dying. To identify the proteins involved in this process, we conducted a comparative proteomic analysis. Protein expression changes among the three different cell types were examined by two-dimensional gel electrophoresis. Among approximately 1000 reproducibly detected protein spots on each gel, 43 were down-regulated and 34 were up-regulated in PCD process. Mass spectrometry identified 17 differentially expressed proteins, including some well-studied proteins as well as some novel PCD related proteins, such as caspases, proteasome subunit, elongation factor, heat shock protein, and hypothetical proteins. Our results suggest that these proteins may participate in the silk gland PCD process of B. mori and, thus, provide new insights for this mechanism.     

Death commitment in the anterior silk gland of the silkworm, Bombyx mori

The insect steroid hormone, 20-hydroxyecdysone (20E) triggers the programmed cell death (PCD) of the anterior silk glands (ASGs) of the silkworm, Bombyx mori. We tried to determine the time of commitment to die (death commitment) by examining ASG responses to 20E and juvenile hormone analogue (JHA) in vivo as well as in vitro. The ASGs obtained late on day 6 of the fifth instar completed PCD when cultured with 20E, while the ASGs obtained on day 4 and cultured with 20E did not undergo PCD. The ASGs became competent to respond to 20E at mid-day 5. The ASGs with responsiveness to 20E were not sensitive to JHA, indicating that the ASGs were committed to die before becoming capable of responding to 20E. Topical application of JHA on day 4 suppressed 20E-induced PCD, but that on day 5 failed to do so, indicating that the death commitment might occur between day 4 and 5. We also determined the time of death commitment after allatectomy of the fourth instar larvae, a procedure that induced the precocious PCD. Timed application of JHA and culture of ASGs with 20E in the presence of JHA showed that the ASGs had lost their sensitivity to JHA between 72 and 96 h after allatectomy, i.e. 24-48 h before precocious gut purge in the allatectomized larvae. This result is similar to that obtained in the fifth instar. We conclude that the cellular commitment to die takes place one day before the ASGs become competent to respond to 20E.     

Ecdysteroid-induced programmed cell death and cell proliferation during pupal wing development of the silkworm, Bombyx mori

The wing margin of adult wings of Lepidoptera is defined by the position of a "bordering lacuna"(BL). During adult wing development, cell proliferation and scale formation proximal to this lacuna and programmed cell death distal to the lacuna are generally observed. To determine the effect of 20-hydroxyecdysone (20E) on these events, we cultured the silkworm pupal wings with or without 20E and analyzed regional specificity for cell death by the TUNEL method and cell proliferation by 5-bromodeoxyuridine labeling. Programmed cell death was induced by 20E after 5 days of culture and was detected only in the region distal to BL. Cell proliferation after 1 day of culture and scale formation after 5 days of culture were also inducible by 20E and detected in the region proximal to BL. These results suggest that two types of pupal wing cells, which are divided by the position of the BL, respond to ecdysteroid in different manners. Higher concentrations of 20E (more than 1,000 ng/ml) repressed the scale formation, while such repression could not be observed in the peripheral cell death even with 5,000 ng/ml 20E. The ecdysteroid may work both as a trigger to make the wing margin and scales and as a developmental timer to arrange these cellular responses.     

Developmental profile of annexin IX and its possible role in programmed cell death of the Bombyx mori anterior silk gland

During pupal metamorphosis, the anterior silk gland (ASG) of the silkworm, Bombyx mori, undergoes programmed cell death (PCD), which is triggered by 20-hydroxyecdysone (20E). Annexin IX (ANX IX) has been identified as a 20E-inducible gene in dying ASGs, and we show here that its expression is down-regulated in tissues destined to die but not in tissues that survive pupal metamorphosis. ANX IX expression was high in the ASGs during the feeding period, when the ecdysteroid titer was low, and decreased in response to the rising ecdysteroid titer that triggered pupal metamorphosis. Before gut purge, in vitro exposure of the ASGs to 20E levels corresponding to the ecdysteroid concentration present at the time of gut purge caused a decrease in ANX IX messenger RNA levels. Expression profiles of EcR and USP, and the 20E concentration-responses of these genes, indicate the importance of the relative abundance of EcR-A and EcR-B1 isoforms in ANX IX regulation. These results suggest an involvement of ANX IX in the determination of PCD timing by delaying or suppressing the response to the increase in hemolymph ecdysteroid concentration during the prepupal period.     

A rapid increase in cAMP in response to 20-hydroxyecdysone in the anterior silk glands of the silkworm, Bombyx mori

In the anterior silk glands (ASGs) of the silkworm, Bombyx mori, intracellular cAMP increases transiently to a very high level shortly after the hemolymph ecdysteroid peak in the prepupal period. In cultured ASGs obtained on the day of gut-purge, cAMP levels were increased by 20-hydroxyecdysone (20E), and this increase was enhanced by an inhibitor of phosphodiesterase, but was not affected by alpha-amanitin, indicating the 20E action may not be mediated via gene expression. The increase in cAMP occurred within 30 seconds of exposure to a physiological concentration of 20E (1 microM), and also by ponasterone A. Our findings indicate a nongenomic action of ecdysteroids in insects, which may be an additional mechanism by which this steroid hormone induces acute responses in tissues and cells.     

Intracellular mobilization of Ca by the insect steroid hormone 20-hydroxyecdysone during programmed cell death in silkworm anterior silk glands

20-Hydroxyecdysone (20E) triggers programmed cell death (PCD) and regulates de novo gene expression in the anterior silk glands (ASGs) of the silkworm Bombyx mori. PCD is mediated via a nongenomic pathway that includes Ca²⁺ as a second messenger and the activation of protein kinase C/caspase-3-like protease; however, the steps leading to a concomitant buildup of intracellular Ca²⁺ are unknown. We employed pharmacological tools to identify the components of this pathway. ASGs were cultured in the presence of 1 μM 20E and one of the following inhibitors: a G-protein-coupled receptor (GPCR) inhibitor, a phospholipase C (PLC) inhibitor, an inositol 1,4,5-trisphosphate receptor (IP₃R) antagonist, and an L- or T-type Ca²⁺ channel blocker. The T-type Ca²⁺ channel blocker inhibited 20E-induced nuclear and DNA fragmentation; in contrast, PCD was induced by 20E in Ca²⁺-free medium, indicating that the source of Ca²⁺ is an intracellular reservoir. The IP₃R antagonist inhibited nuclear and DNA fragmentation, suggesting that the endoplasmic reticulum may be the Ca²⁺ source. Finally, the GPCR and PLC inhibitors effectively blocked nuclear and DNA fragmentation. Our results indicate that 20E increases the intracellular level of Ca²⁺ by activating IP₃R, and that this effect may be brought about by the serial activation of GPCR, PLC, and IP₃.     

Solubilization of the ecdysone binding protein from anterior silk gland cell membranes of the silkworm, Bombyx mori

We previously provided preliminary evidence for the presence of a putative membrane ecdysone receptor (mEcR) anchored in the plasma membranes of anterior silk glands (ASGs) in Bombyx mori. This receptor may act in concert with the conventional EcR in 20E-dependent programmed cell death of these glands. We report here, for the first time, the solubilization of mEcR from ASG membranes using the zwitterionic detergent CHAPS in the presence of NaCl. Our results show by ligand binding assay that mEcR solubilized this way is functionally active and retains 75% of its native binding activity. We also defined experimental conditions that yielded protein/detergent complexes with partial binding activity, which makes it possible to purify the membrane-bound ecdysone binding protein.     

Soluble and particle-bound trehalase in the silk glands during larval-pupal development of the silkworm, Bombyx mori

Trehalase localization in silk glands of the silkworm, Bombyx mori has been studied during larval-pupal development. Subcellular distribution of the silk gland trehalase depends upon larval-pupal development. The activity increases in the soluble fraction with a concomitant decrease in the particle-bound fraction during larval-pupal development. The pH-optimum value of trehalase activity in the particle-bound fraction changes from 6.5 to 6.0, and in the soluble fraction from 5.5 to 4.5 in the course of the silk gland degeneration during metamorphosis.     

RNA干扰对家蚕丝腺蜕皮激素相关基因表达的影响

昆虫变态发育过程中,蜕皮激素通过一系列的激素相关转录因子进行信号的转导和放大,从而完成对生长变态发育的调控,其中蜕皮激素受体(EcR)及转录因子BR-C和E74A可能作为早期因子发挥作用.为了研究这3个早期转录因子在鳞翅目昆虫中的功能,本研究采用体外合成dsRNA的方法,将合成的dsRNA分别注射熟蚕期的家蚕Bomby...     

Studies on silk fibroin of the silkworm Bombyx mori

A procedure has been developed to obtain native fibroin in a pure state from the reservoir part of the silk gland. The purified protein has a sedimentation coefficient of 10 S as determined on sucrose density gradients and the amino acid composition is similar to that reported for fibroin from the cocoons. The effects of various solvents has been studied; lithium thiocyanate was found to be the solvent of choice. By in vivo labeling of fibroin with [³H]glycine and [¹⁴C]alanine it was demonstrated that fibroin synthesized in the posterior part of the gland and that stored in the reservoir part are identical.     

Copper in the silk formation process of Bombyx mori silkworm

Evidence is presented here that cupric ions play a part in the natural spinning of Bombyx mori silk. Proton induced X-ray emission studies revealed that the copper content increased from the posterior part to the anterior part of silk gland, and then further increased in the silk fiber. Spectrophotometric analysis demonstrated that cupric ions formed coordination complexes with silk fibroin chains while Raman spectroscopy indicated that they induced a conformation transition from random coil/helix to beta-sheet. Taken together these findings indicate that copper could play a role in the natural spinning process in silkworms.     

Glucose oxidase prevents programmed cell death of the silkworm anterior silk gland through hydrogen peroxide production

During pupal metamorphosis, the anterior silk glands (ASGs) of the silkworm Bombyx mori degenerate through programmed cell death (PCD), which is triggered by 20-hydroxyecdysone (20E). 20E triggers the PCD of the ASGs of day 7 fifth instar (V7) larvae but not that of V5 larvae. When V7 ASGs were cocultured with V5 ASGs in the presence of 20E, neither culture of ASGs underwent PCD. The 20E-induced PCD of V7 ASGs was also inhibited when they were incubated in conditioned medium that was prepared by incubating V5 ASGs for 48 h, an indication that V5 ASGs released an inhibitor of 20E-induced PCD during incubation. The inhibitor was purified from conditioned medium and identified as glucose oxidase (GOD). GOD catalyzes the oxidation of glucose to gluconolactone, and generates hydrogen peroxide as a byproduct. We found that hydrogen peroxide is the molecule that directly inhibits the action of 20E and may act to protect the ASGs from early execution of PCD during the feeding stage. GOD was localized in the inner cavity of the gland, and was discharged to the outside of the ASGs with the silk thread at the onset of spinning. Thus, the spinning behavior, occurring at the beginning of the prepupal period, plays an important role in controlling the time at which ASGs undergo PCD in response to 20E.     

Presence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx mori.

Nongenomic action of an insect steroid hormone, 20-hydroxyecdysone (20E), has been implicated in several 20E-dependent events including the programmed cell death of Bombyx anterior silk glands (ASGs), but no information is available for the mode of the action. We provide evidence for a putative membrane receptor located in the plasma membrane of the ASGs. Membrane fractions prepared from the ASGs exhibit high binding activity to [3H]ponasterone A (PonA). The membrane fractions did not contain conventional ecdysone receptor as revealed by Western blot analysis using antibody raised against Bombyx ecdysone receptor A (EcR-A). The binding activity was not solubilized with 1 m NaCl or 0.05% (w/v) MEGA-8, indicating that the binding sites were localized in the membrane. Differential solubilization and temperature-induced phase separation in Triton X-114 showed that the binding sites might be integrated membrane proteins. These results indicated that the binding sites are located in plasma membrane proteins, which we putatively referred to as membrane ecdysone receptor (mEcR). The mEcR exhibited saturable binding for [3H]PonA (Kd = 17.3 nm, Bmax = 0.82 pmol.mg(-1) protein). Association and dissociation kinetics revealed that [3H]PonA associated with and dissociated from mEcR within minutes. The combined results support the existence of a plasmalemmal ecdysteroid receptor, which may act in concert with the conventional EcR in various 20E-dependent developmental events.     

Proteome analysis of silk gland proteins from the silkworm, Bombyx mori

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.     

Insulin stimulates ecdysteroidogenesis by prothoracic glands in the silkworm,Bombyx mori

It is generally accepted that the prothoracicotropic hormone (PTTH) is the stimulator of ecdysteroidogenesis by prothoracic glands in larval insects. In the present study, we investigated activation of ecdysteroidogenesis by bovine insulin in prothoracic glands of the silkworm, Bombyx mori. The results showed that the insulin stimulated ecdysteroidogenesis during a long-term incubation period and in a dose-dependent manner. In addition, insulin also stimulated both DNA synthesis and viability of prothoracic glands. Insulin-stimulated ecdysteroidogenesis was blocked by either LY294002 or wortmannin, indicating involvement of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Activation of ecdysteroidogenesis by insulin appeared to be developmentally regulated. Moreover, in vitro activation of ecdysteroidogenesis of prothoracic glands by insulin was also verified by in vivo experiments: injection of insulin into day 6 last instar larvae greatly increased both hemolymph ecdysteroid levels and ecdysteroidogenesis 24 h after the injection, indicating its possible in vivo function. Phosphorylation of Akt and the insulin receptor was stimulated by insulin, and stimulation of Akt phosphorylation appeared to be PI3K-dependent and developmentally regulated. Insulin did not stimulate extracellular signal-regulated kinase (ERK) signaling of the prothoracic glands. These results suggest that in silkworm prothoracic glands, in addition to the PTTH and an autocrine factor, ecdysteroidogenesis is also stimulated by insulin during development.     

DNA polymerase-delta from the silk glands of Bombyx mori.

The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3'----5' exonuclease activity which participates in proofreading by mismatch repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated.