The Krüppel homolog 1 gene (Kr-h1) has been proposed to play a key role in the repression of insect metamorphosis. Kr-h1 is assumed to be induced by juvenile hormone (JH) via a JH receptor, methoprene-tolerant (Met), but the mechanism of induction is unclear. To elucidate the molecular mechanism of Kr-h1 induction, we first cloned cDNAs encoding Kr-h1 (BmKr-h1) and Met (BmMet1 and BmMet2) homologs from Bombyx mori. In a B. mori cell line, BmKrh1 was rapidly induced by subnanomolar levels of natural JHs. Reporter assays identified a JH response element (kJHRE), comprising 141 nucleotides, located ∼2 kb upstream from the BmKr-h1 transcription start site. The core region of kJHRE (GGCCTCCACGTG) contains a canonical E-box sequence to which Met, a basic helix-loophelix Per-ARNT-Sim (bHLH-PAS) transcription factor, is likely to bind. In mammalian HEK293 cells, which lack an intrinsic JH receptor, ectopic expression of BmMet2 fused with Gal4DBD induced JHdependent activity of an upstream activation sequence reporter. Meanwhile, the kJHRE reporter was activated JH-dependently in HEK293 cells only when cotransfected with BmMet2 and BmSRC, another bHLH-PAS family member, suggesting that BmMet2 and BmSRC jointly interact with kJHRE. We also found that the interaction between BmMet2 and BmSRC is dependent on JH. Therefore, we propose the following hypothesis for the mechanism of JHmediated induction of BmKr-h1: BmMet2 accepts JH as a ligand, JH-liganded BmMet2 interacts with BmSRC, and the JH/BmMet2/ BmSRC complex activates BmKr-h1 by interacting with kJHRE. development | insecticide | steroid receptor coactivator

Previously, we isolated a cDNA clone for the ecdysone receptor B1 isoform of the silkworm, Bombyx mori (BmEcR-Bl). Here we report the cloning of a cDNA that encodes the Bombyx ecdysone receptor A isoform (BmEcR-A) and mRNA expression of the two BmEcR isoforms during molting and metamorphosis. At larval-pupal transformation, mRNA expression of BmEcR-B 1 was predominant in most tissues examined, including three larval tissues (midgut, epidermis, and fat body) and the wing imaginal disc. The anterior silk gland was the only tissue where BmEcR-A was predominant. These expression patterns were different from observations demonstrated in Drosophila. In the anterior silk gland, both EcR isoforms were expressed synchronously during the fifth larval instar, while expression of the A isoform preceded that of the B1 isoform by two days in the fourth instar. Precedence of BmEcR-A during the fourth instar and synchronization of both isoforms during the fifth instar were also observed in the middle and posterior silk glands, suggesting that transcription of BmEcR in the silk gland is regulated differently in these two instars. In the cultured anterior silk glands of day 0 of the fifth instar, transcription of BmEcR-A and BmEcR-B1 was induced dose dependently by more than 5 ng/ml 20-hydroxyecdysone. BmEcR-A and BmEcR-B1 mRNAs were induced within 2 h and 1 h, respectively, of the addition of 20-hydroxyecdysone. These results suggest that the increase of BmEcR mRNAs during the fifth instar is induced in vivo by a small increase in ecdysteroids.Keywords: ecdysone receptor; Bombyx mori; ecdysteroid ; silk gland ; isoform.Steroid hormones coordinate a wide array of developmental and physiological processes in higher organisms, through binding with receptor proteins to regulate the stage-specific and tissue-specific transcription of target genes. In insects, ecdysteroids, particularly 20-hydroxyecdysone, are the key steroid hormones. 20-Hydroxyecdysone plays a central role in the orchestration of development during molts and metamorphosis. Ashburner et al. (1974) proposed a model for 20-hydroxyecdysone action based on puffing responses of the polytene chromosomes in the Drosophila salivary gland. In this model, 20-hydroxyecdysone binds with an ecdysone receptor protein and directly activates the transcription of a small set of early genes, which then activate many late genes. Recent molecular analyses characterized the ecdysone receptor gene and some of the early genes, most of which are putative transcriptional factors as predicted by Ashburner's model (reviewed in Henrich and Brown, 1995;Thummel, 1995). The ecdysone receptor (EcR) identified from Drosophila is a member of the nuclear receptor superfamily and has three isoforms (A, B1, and B2) with common DNA and

The insect cytokine paralytic peptide (PP) induces muscle contraction in silkworm larvae. Here we demonstrate that bacterial and fungal cell wall components peptidoglycan and glucan stimulate muscle contraction via activation of PP in the hemolymph. Anti-PP antibody suppressed the muscle contraction induced by PP, peptidoglycan, or glucan. The contraction was also inhibited by free radical scavengers and serine protease inhibitors. Moreover, injecting live silkworms with peptidoglycan or glucan generated the active form of PP. The active form of PP was also produced in vitro when peptidoglycan or glucan was incubated with hemolymph containing the PP precursor. Generation of the active form of PP was suppressed by free radical scavengers and serine protease inhibitors. Furthermore, PP activation in isolated hemolymph was inhibited by potassium cyanide, suggesting that cellular activity is involved. Stimulation by peptidoglycan promoted the generation of reactive oxygen species by silkworm hemocytes. The addition of either the active form of PP or anti-PP antibody to Staphylococcus aureus injected into silkworm larvae delayed or enhanced, respectively, the killing effect of S. aureus, suggesting that activated PP contributes to host resistance to infectious pathogens. These findings suggest that immunologic stimulants such as peptidoglycan or glucan induce reactive oxygen species production from larval hemocytes, followed by the activation of serine protease, which mediates the PP processing reaction and leads to defensive responses.

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In the blood (hemolymph) of the silkworm Bombyx mori, the insect cytokine paralytic peptide (PP) is converted from an inactive precursor to an active form in response to the cell wall components of microorganisms and contributes to silkworm resistance to infection. To investigate the molecular mechanism underlying the up-regulation of host resistance induced by PP, we performed an oligonucleotide microarray analysis on RNA of blood cells (hemocytes) and fat body tissues of silkworm larvae injected with active PP. Expression levels of a large number of immune-related genes increased rapidly within 3 h after injecting active PP, including phagocytosis-related genes such as tetraspanin E, actin A1, and ced-6 in hemocytes, and antimicrobial peptide genes cecropin A and moricin in the fat body. Active PP promoted in vitro and in vivo phagocytosis of Staphyloccocus aureus by the hemocytes. Moreover, active PP induced in vivo phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) in the fat body. Pretreatment of silkworm larvae with ML3403, a pharmacologic p38 MAPK inhibitor, suppressed the PP-dependent induction of cecropin A and moricin genes in the fat body. Injection of active PP delayed the killing of silkworm larvae by S. aureus, whereas its effect was abolished by preinjection of the p38 MAPK inhibitor, suggesting that p38 MAPK activation is required for PP-dependent defensive responses. These findings suggest that PP acts on multiple tissues in silkworm larvae and acutely activates cellular and humoral immune responses, leading to host protection against infection.