Silkworm Cells Research Articles

Role of Bmbuffy in hydroxycamptothecine-induced apoptosis in BmN-SWU1 cells of the silkworm, Bombyx mori

Bcl-2 family proteins have been reported previously to play important roles in the mitochondrial apoptotic pathway. Particularly, Bmbuffy has been identified as a key homologue of Bcl-2 in silkworm; however, its exact function is unknown. In this study, we investigated the role of Bmbuffy in hydroxycamptothecine (HCPT)-induced apoptosis of BmN-SWU1 cells. By conducting confocal microscopy studies, we found that Bmbuffy is located on the outer membrane of mitochondria and endoplasmic reticulum (ER). Furthermore, we discovered that the hydrophobic transmembrane domain at the COOH terminus is a putative anchor for the subcellular localization of Bmbuffy. Overexpression of Bmbuffy inhibited cytochrome c release, activation of caspase-3 and cell apoptosis, while RNAi-mediated silencing of Bmbuffy promoted apoptosis. In the absence of a hydrophobic membrane anchor, we revealed that Bmbuffy is unable to block apoptosis. These results indicate that Bmbuffy acts as an anti-apoptotic protein, located on the mitochondrial outer membrane and is involved in the mitochondrial apoptotic pathway. Moreover, in HCPT-induced apoptosis, we showed that the translocation of endogenous Bmp53 from the nucleus to the mitochondria is a slow and progressive process, followed by cytochrome c release. This suggests that mitochondrial Bmp53 accumulation may contribute to membrane permeability. The co-localization of Bmp53 and Bmbuffy suggests the interaction of the two proteins, which was further confirmed by Co-IP assay. In addition, overexpression of Bmp53 increased cytochrome c release and the cell apoptotic rate, whereas Bmbuffy overexpression blocked these. All the data suggest that Bmbuffy functions as an anti-apoptotic protein and interacts with Bmp53 in HCPT-induced apoptosis of silkworm cells.

Roles of Piwi Proteins in Transcriptional Regulation Mediated by HP1s in Cultured Silkworm Cells

Piwi proteins are part of a superfamily of Argonaute proteins, which are one of the core components of the RNA silencing pathway in many eukaryotes. Piwi proteins are thought to repress the transposon expression both transcriptionally and post-transcriptionally. Recently, Drosophila melanogaster Piwi was recently reported to associate with chromatin and to interact directly with the Heterochromatin Protein 1 (HP1a). However, similar interactions have not been reported in other higher eukaryotes. Here we show that silkworm Piwi proteins interact with HP1s in the nucleus. The silkworm, Bombyx mori, has two Piwi proteins, Ago3 and Siwi, and two typical HP1 proteins, HP1a and HP1b. We found that HP1a plays an important role in the interaction between Ago3/Siwi and HP1b in the ovary-derived BmN4 cell line. We also found that Ago3/Siwi regulates the transcription in an HP1-dependent manner. These results suggest that silkworm Piwi proteins function as a chromatin regulator in collaboration with HP1a and HP1b.

Niemann–Pick disease type C2 protein induces triglyceride accumulation in silkworm and mammalian cell lines

Silkworm haemolymph induced both the cessation of growth and an increase in triglyceride (triacylglycerol) storage in BmN4 cells. We purified the growth inhibitory factor from the silkworm haemolymph and identified this protein as the Bombyx mori PP (promoting protein), an orthologue of NPC2 (Niemann-Pick disease type C2) protein. Recombinant silkworm NPC2 inhibited cellular proliferation and increased triglyceride accumulation in BmN4 cells. Injection of either the recombinant protein or antiserum of NPC2 into living silkworms increased or decreased respectively triglyceride levels in the fat body. A mutation that depletes the cholesterol-binding capacity did not abolish the activity of NPC2. We further revealed that NPC2 induced the phosphorylation of AMPK (AMP-activated protein kinase) and that an AMPK inhibitor suppressed NPC2-dependent triglyceride accumulation. These findings suggest that NPC2 induces triglyceride accumulation via the activation of AMPK independently of its cholesterol-binding capacity in the silkworm.

Inhibition of BmNPV replication in silkworm cells using inducible and regulated artificial microRNA precursors targeting the essential viral gene lef-11

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major silkworm pathogen, causing substantial economic losses to the sericulture industry annually. We demonstrate a novel anti-BmNPV system expressing mature artificial microRNAs (amiRNAs) targeting the viral lef-11 gene. The mature amiRNAs inhibited the lef-11 gene in silkworm BmN-SWU1 cells. Antiviral assays demonstrated that mature amiRNAs silenced the gene and inhibited BmNPV proliferation efficiently. As constitutive overexpression of mature amiRNAs may induce acute cellular toxicity, we further developed a novel virus-induced amiRNA expression system. The amiRNA cassette is regulated by a baculovirus-induced fusion promoter. This baculovirus-induced RNA interference system is strictly regulated by virus infection, which functions in a negative feedback loop to activate the expression of mature amiRNAs against lef-11 and subsequently control inhibition of BmNPV replication. Our study advances the use of a regulatable amiRNA cassette as a safe and effective tool for research of basic insect biology and antiviral application.

Molecular cloning and characterization of a recombinant Bombyx mori tyramine-β-hydroxylase in a silkworm cell line using a baculovirus expression vector system

Octopamine (OA) and tyramine (TA) are biogenic amines that act as neurotransmitters, neurohormones, and neuromodulators in the invertebrate nervous system. Tyramine-β-hydroxylase (TβH) catalyzes the biosynthesis of OA from TA. In this study, cDNA encoding Bombyx mori TβH (BmTβH) was cloned from the brain of the silkworm B. mori. The BmTβH mRNA comprised 2204 nucleotide residues and contained an open reading frame encoding 592 amino acids. The deduced amino acid sequence shared homology to several proteins belonging to the insect TβH family. Functional expression of the cloned cDNA was obtained using a B. mori baculovirus expression vector system. Western blot analysis revealed an immunoreactive band with a molecular mass of ~67.4kDa. Reverse-phase high-performance liquid chromatography (HPLC) was used to identify the products formed during incubation of the enzyme reaction mixture. The optimum pH and temperature for the conversion of TA to OA were 7.5 and 25°C, respectively. During incubation, the reaction was linear for the first 30min at 25°C and pH7.5. Inhibitory experiments carried out with various concentrations of an inhibitor showed that this method can be used for screening of BmTβH inhibitors.

Chromatin-induced spindle assembly plays an important role in metaphase congression of silkworm holocentric chromosomes

The kinetochore plays important roles in cell cycle progression. Interactions between chromosomes and spindle microtubules allow chromosomes to congress to the middle of the cell and to segregate the sister chromatids into daughter cells in mitosis. The chromosome passenger complex (CPC), composed of the Aurora B kinase and its regulatory subunits INCENP, Survivin, and Borealin, plays multiple roles in these chromosomal events. In the genome of the silkworm, Bombyx mori, which has holocentric chromosomes, the CPC components and their molecular interactions were highly conserved. In contrast to monocentric species, however, the silkworm CPC co-localized with the chromatin-driven spindles on the upper side of prometaphase chromosomes without forming bipolar mitotic spindles. Depletion of the CPC by RNAi arrested the cell cycle progression at prometaphase and disrupted the microtubule network of the chromatin-driven spindles. Interestingly, depletion of mitotic centromere-associated kinesin (MCAK) recovered formation of the microtubule network but did not overcome the cell cycle arrest at prometaphase. These results suggest that the CPC modulates the chromatin-induced spindle assembly and metaphase congression of silkworm holocentric chromosomes.

Cloning, Expression Feature, and Tissue Location of BmSCRBQ1, a Class B Scavenger Receptor in Bombyx mori (L.)

Abstract Class B scavenger receptors can combine with various kinds of ligands and are involved in lipid metabolism. They play an important role in a series of physiological processes, such as atherosclerosis formation or inhibition, congenital immune defense, apoptotic cell clearance, carotenoid transport, and pheromone transduction. In this study, based on a whole-genome database of the silkworm (Bombyx mori (L.)), the full-length CDS of BmSCRBQ1, a class B scavenger receptor gene, was cloned. BmSCRBQ1 consists of 10 exons and 9 introns, with an open reading frame of 1,482 bp encoding 493 amino acids. BmSCRBQ1 mRNA and protein were expressed in most tissues of larval B. mori strain Dazao and the silkworm cell lines BmN and BmE, suggesting that BmSCRBQ1 may have certain ubiquitous functions. Immunohistochemical staining showed that BmSCRBQ1 protein is located in the constitutive membranes or cell membranes of the testes, ovaries, fat body, blood cells, and the middle silk gland of larval B. mori Dazao, indicating that its function could be related to lipid metabolism; the binding, transport, and phagocytosis of exogenous materials; and signal transduction in these tissue types.

Protective Effect of the Silkworm Protein 30Kc6 on Human Vascular Endothelial Cells Damaged by Oxidized Low Density Lipoprotein (Ox-LDL)

Although the 30K family proteins are important anti-apoptotic molecules in silkworm hemolymph, the underlying mechanism remains to be investigated. This is especially the case in human vascular endothelial cells (HUVECs). In this study, a 30K protein, 30Kc6, was successfully expressed and purified using the Bac-to-Bac baculovirus expression system in silkworm cells. Furthermore, the 30Kc6 expressed in Escherichia coli was used to generate a polyclonal antibody. Western blot analysis revealed that the antibody could react specifically with the purified 30Kc6 expressed in silkworm cells. The In vitro cell apoptosis model of HUVEC that was induced by oxidized low density lipoprotein (Ox-LDL) and in vivo atherosclerosis rabbit model were constructed and were employed to analyze the protective effects of the silkworm protein 30Kc6 on these models. The results demonstrated that the silkworm protein 30Kc6 significantly enhanced the cell viability in HUVEC cells treated with Ox-LDL, decreased the degree of DNA fragmentation and markedly reduced the level of 8-isoprostane. This could be indicative of the silkworm protein 30Kc6 antagonizing the Ox-LDL-induced cell apoptosis by inhibiting the intracellular reactive oxygen species (ROS) generation. Furthermore, Ox-LDL activated the cell mitogen activated protein kinases (MAPK), especially JNK and p38. As demonstrated with Western analysis, 30Kc6 inhibited Ox-LDL-induced cell apoptosis in HUVEC cells by preventing the MAPK signaling pathways. In vivo data have demonstrated that oral feeding of the silkworm protein 30Kc6 dramatically improved the conditions of the atherosclerotic rabbits by decreasing serum levels of total triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC). Furthermore, 30Kc6 alleviated the extent of lesions in aorta and liver in the atherosclerotic rabbits. These data are not only helpful in understanding the anti-apoptotic mechanism of the 30K family proteins, but also provide important information on prevention and treatment of human cardiovascular diseases.

V-ATPase Is Involved in Silkworm Defense Response against Bombyx mori Nucleopolyhedrovirus

Silkworms are usually susceptible to the infection of Bombyx mori (B. mori) nucleopolyhedrovirus (BmNPV), which can cause significant economic loss. However, some silkworm strains are identified to be highly resistant to BmNPV. To explore the silkworm genes involved in this resistance in the present study, we performed comparative real-time PCR, ATPase assay, over-expression and sub-cellular localization experiments. We found that when inoculated with BmNPV both the expression and activity of V-ATPase were significantly up-regulated in the midgut column cells (not the goblet cells) of BmNPV-resistant strains (NB and BC8), the main sites for the first step of BmNPV invasion, but not in those of a BmNPV-susceptible strain 306. Furthermore, this up-regulation mainly took place during the first 24 hours post inoculation (hpi), the essential period required for establishment of virus infection, and then was down-regulated to normal levels. Amazingly, transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm cells could significantly inhibit BmNPV proliferation. To our knowledge this is the first report demonstrating clearly that V-ATPase is indeed involved in the defense response against BmNPV. Our data further suggests that prompt and potent regulation of V-ATPase may be essential for execution of this response, which may enable fast acidification of endosomes and/or lysosomes to render them competent for degradation of invading viruses.

Expression of freeze‐responsive proteins, Fr10 and Li16, from freeze‐tolerant frogs enhances freezing survival of BmN insect cells

To date, two novel freeze-responsive proteins, Fr10 and Li16, have been discovered in the wood frog, Rana sylvatica, and likely support freezing survival. Although previous studies have established tissue distribution of each protein, there have been no studies that explore their functional consequences in intolerant cells. To assess the ability of Fr10 and Li16 to confer freeze tolerance, we transfected each protein into a freeze-intolerant silkworm cell line (BmN). Selected controls were the transfection of an unrelated protein (CAT) and a no-transfection sample. Li16 and Fr10 showed 1.8 ± 0.1- and 1.7 ± 0.2-fold, respectively, greater survival after freezing at -6°C for 1 h than did transfection controls. To investigate how these novel proteins protect cells from freezing damage, protein structures were predicted from primary amino acid sequences. Analysis of the structures indicated that Fr10 is a secreted protein and may be a new type IV antifreeze protein, whereas Li16 may have intracellular membrane associated functions. This study shows that freezing protection can be provided to intolerant cells by the overexpression of transfected Li16 and Fr10 frog proteins. Results from this study will provide new insights into adapting intolerant cells for medical organ cryoprotection using a natural vertebrate model of tolerance.

AMINO ACID DEPRIVATION‐INDUCED EXPRESSION OF ASPARAGINE SYNTHETASE REGULATES THE GROWTH AND SURVIVAL OF CULTURED SILKWORM CELLS

Expression of Bombyx mori Asparagine synthetase (BmASNS), one gene that encodes an enzyme catalyzing asparagine biosynthesis, is transcriptionally induced following amino acid deprivation. Previous transcriptional analysis of the BmASNS gene showed the involvement of Polycomb proteins, epigenetic repressors, in suppressing BmASNS expression in a cell cycle-dependent manner. However, the role of BmAsns protein in these cellular processes remains unclear. The present study thus exploited the potential function of BmAsns protein in cultured silkworm cells. Our results showed that ectopic overexpression of BmASNS gene effectively inhibited cell growth in silkworm cells, whereas its overexpression could rescue cell growth upon amino acid deprivation treatment. We found that the cells expressing BmAsns protein were capable of influencing the formation of autophagic vacuoles stimulated by amino acid deprivation. We speculated that the recovery of cell growth by overexpressed BmAsns protein is due to the rapid turnover of autophagic vacuoles in the cells. To further assess the effects of BmAsns on cell development, we used RNA interference to silence BmASNS expression in silkworm cells in the presence or absence of amino acids. Our results revealed a significant change of cell proliferation as well as cell cycle distribution after knockdown of BmASNS. Importantly, silkworm cells lacking BmASNS under the condition of amino acid deprivation showed severely impaired proliferation. Altogether, we concluded that the up-regulated expression of BmASNS would be able to protect cells from impairment induced by amino acid deprivation, which in turn facilitates cell growth and survival.

RNAi suppression of β-N-acetylglucosaminidase (BmFDL) for complex-type N-linked glycan synthesis in cultured silkworm cells

Glycoproteins have various biological functions including enzymatic activity, protein stability and others. Due to the presence of paucimannosidic N-linked glycans, recombinant proteins from an insect cell expression system may not be suitable for therapeutic use. Because baculovirus expression systems (BESs) are used to produce recombinant proteins, it is of interest to modify the endogenous N-glycosylation pathway in insects to mimic that of mammals. Using a soaking RNAi sensitive cell line, BmN4-SID1, has enabled us to suppress Bombyx mori FDL (BmFDL), an N-linked glycan-specific β-N-acetylglucosaminidase. Western blotting and MALDI-TOF MS demonstrated that the BmFDL depletion almost completely converted the paucimannosidic structures of the recombinant proteins produced by BES into a complex-type structure. This highly efficient, simple and low-cost method can be used for mass production of secretion proteins with complex-type N-linked glycans.

BmCyclin B and BmCyclin B3 are required for cell cycle progression in the silkworm, Bombyx mori

Cyclin B is an important regulator of the cell cycle G2 to M phase transition. The silkworm genomic database shows that there are two Cyclin B genes in the silkworm (Bombyx mori), BmCyclin B and BmCyclin B3. Using silkworm EST data, the cyclin B3 (EU074796) gene was cloned. Its complete cDNA was 1665 bp with an ORF of 1536 bp derived from seven exons and six introns. The BmCyclin B3 gene encodes 511 amino acids, and the predicted molecular weight is 57.8 kD with an isoelectric point of 9.18. The protein contains one protein damage box and two cyclin boxes. RNA interference-mediated reduction of BmCyclin B and BmCyclin B3 expression induced cell cycle arrest in G2 or M phase in BmN-SWU1 cells, thus inhibiting cell proliferation. These results suggest that BmCyclin B and BmCyclin B3 are necessary for completing the cell cycle in silkworm cells.

Construction of the ie1-Bacmid Expression System and Its Use to Express EGFP and BmAGO2 in BmN Cells

The presently available expression tools and vectors (e.g., eukaryotic expression vectors and the adenovirus expression system) for studying the functional genes in Bombyx mori are insufficient. The baculovirus expression system is only used as a protein production tool; therefore, recombinant proteins expressed by B. mori using the baculovirus expression system equipped with a polyhedrin promoter cannot be used for in vivo research applications. In this work, we constructed and screened a eukaryotic expression vector for silkworm cells The EGFP and B. mori Argonaute2 proteins were found to be efficiently expressed using the screened pIEx-1 vector with the FuGENE 6 transfection reagent. Additionally, we constructed a novel nucleopolyhedrovirus ie1-Bacmid expression system for the production of recombinant protein; we then used the system to highly express the EGFP and B. mori Argonaute2 proteins. In this system, the protein of interest can be efficiently expressed 13h after infection by controlling the B. mori nucleopolyhedrovirus immediate early ie1 promoter. The ie1-Bacmid system provides a powerful "adenovirus-like" expression tool; not only can the tool be used to study baculovirus molecular biology for the silkworm but it is also useful in other research applications as well, such as the study of gene functions involved in cellular physiological processes.

The three G’s

It was my good fortune to meet personally the three invertebrate cell culture pioneers, Richard Goldschmidt, Zan-Yin Gaw, and Thomas D. C. Grace (Fig. 1). In 1951 I met Goldschmidt at a symposium in Cold Spring Harbor, but I only knew that he was a prominent geneticist. I had no idea about his insect cell culture work at Yale University and daily contacts with Ross G. Harrison. In 1959 Zan Yin Gaw in Wuhan successfully cultured monolayers of silkworm cells for more than one year. I reported his breakthrough achievement at the 11th International Congress of Entomology in Vienna in 1960, but his work was completely ignored outside China. In 1982 Gaw invited me to Wuhan where he told me that he studied in the United States in the 1930s, working as postdoctoral scientist at the Rockefeller Institute, where he was daily meeting William Trager, and later at Yale University in the Osborn Laboratory, where he was inspired by Harrison. T. D. C. Grace worked in my laboratory at Rockefeller University during 1957 and 1958, then returned to CSIRO in Canberra, Australia. In 1962 he successfully established a cell line from the ovarian tissues of Antherea eucalypti pupae. The subsequent expansion of invertebrate cell culture involved the Nobel laureate Rita Levi Montalcini. I met her at a Growth Symposium, where I saw Harrison the last time. In 1969 she published the first of a dozen papers on in vitro studies of the embryonic nerve system of Periplaneta americana that led to her milestone discovery of the nerve growth factors. The “3 G-s” insect cell culture pioneers were directly or indirectly influenced and inspired by Harrison. Gaw was the only one who created a large following of insect cell culture researchers, who continued and expanded the work started by him in China.

Efficient disruption of endogenous Bombyx gene by TAL effector nucleases

Engineered nucleases are proteins that are able to cleave DNA at specified sites in the genome. These proteins have recently been used for gene targeting in a number of organisms. We showed earlier that zinc finger nucleases (ZFNs) can be used for generating gene-specific mutations in Bombyx mori by an error-prone DNA repair process of non-homologous end joining (NHEJ). Here we test the utility of another type of chimeric nuclease based on bacterial TAL effector proteins in order to induce targeted mutations in silkworm DNA. We designed three TAL effector nucleases (TALENs) against the genomic locus BmBLOS2, previously targeted by ZFNs. All three TALENs were able to induce mutations in silkworm germline cells suggesting a higher success rate of this type of chimeric enzyme. The efficiency of two of the tested TALENs was slightly higher than of the successful ZFN used previously. Simple design, high frequency of candidate targeting sites and comparable efficiency of induction of NHEJ mutations make TALENs an important alternative to ZFNs.

Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double-stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome-integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.

Pinellia ternata agglutinin produced in Bombyx mori cells exhibits bioactivity.

Pinellia ternata agglutinin (PTA) is highly homologous to many other monocot mannose-binding lectins which reportedly possess antitumor activities. Its production in silkworm cells has great application potential because the baculovirus expression system can produce post-translationally modified proteins at low cost. In the current study, the pta gene was cloned and expressed in silkworm cells, and the expressed protein was analyzed using a hemagglutination assay. A preliminary in vitro study on its anti-proliferative activity was performed. The results show that the recombinant PTA with an apparent molecular mass of 29 kDa can hemagglutinate rabbit erythrocytes and this activity can be inhibited by D-mannan at a low concentration. In addition, the recombinant hemagglutinin exhibited a dose-dependent anti-proliferative activity on hepatoma cells. The results of the current study suggest that PTA and other important bioactive proteins could be produced by silkworm bioreactor for biomedicine research and application.

Construction of a long hairpin RNA expression library using Cre recombinase

A large number of genome-wide screens using RNA interference (RNAi) libraries have been utilized to determine the function of individual gene products involved in a variety of biological processes. In this study, we describe a new method to enzymatically generate a long hairpin RNA (lhRNA) expression library from a cDNA plasmid library using a nicking endonuclease, BcaBEST DNA polymerase, and Cre recombinase without excising the inserted DNA fragment from the plasmid vector. This method involves 5 steps: (1) conversion of an inserted DNA fragment in a plasmid into a direct repeat (DR); (2) purification of the plasmid containing the DR; (3) subcloning a lox71 cassette into the plasmid; (4) conversion of the DR in the plasmid into an inverted repeat (IR) using Cre recombinase; and (5) purification of the plasmid containing the IR. We also established an efficient method for inserting DNase I-digested DNA fragments into expression plasmids to enable construction of a cDNA plasmid library suitable as source materials to construct the lhRNA expression library. We confirmed that each of the lhRNA expression plasmids constructed using this method induced strong RNAi in a silkworm cell line, NIAS-Bm-oyanagi2.

BmEts upregulates promoter activity of lebocin in Bombyx mori

The Ets family protein BmEts is assumed to be implicated in determination of diapause in the embryogenesis of Bombyx mori. In this study, we found that expression of BmEts was increased in the fat body and other tissues of the 5th instar larvae in response to Escherichia coli injection. Cotransfection experiments using a silkworm cell line revealed that overexpression of BmEts significantly elevated the activity of lebocin promoter but not of cecropin B1, cecropin D, attacin, and moricin promoters. Activation of the lebocin promoter by BmEts was dependent on at least two κB elements and the most proximal GGAA/T motif located on the 5′-upstream region. BmEts further synergistically enhanced E. coli or BmRelish1-d2 (active form)-stimulated lebocin promoter activation. Two κB elements were also found to be involved in promoter activation by BmRelish1-d2 and in synergistic promoter activation by BmEts and BmRelish1-d2 in the silkworm cells. Specific binding of recombinant BmEts to the proximal κB element and the most proximal GGAA/T motif and interaction between BmEts and BmRelish1 were also observed. To our knowledge, this is the first report of an Ets family protein directly regulating immune-related genes in invertebrates.