Takumi Kayukawa¹, Chieka Minakuchi¹, Toshiki Namiki¹, Toru Togawa¹, Michiyo Yoshiyama¹, Manabu Kamimura¹, Kazuei Mita², Makoto Kiuchi¹, Tetsuro Shinoda¹

¹Insect Growth Regulation Research Unit, ²Insect Genome Research Unit

［Abstract］

We clarified the mechanism of juvenile hormone (JH)-mediated induction of Kru¨ppel homolog 1 (Kr-h1), a repressor of insect metamorphosis, using a silkworm (Bombyx mori) cell line. A JH receptor (methoprene tolerant, Met) forms a complex with steroid receptor coactivator (SRC) in the presence of JH, and activates Kr-h1 by interacting with the JH response element of Kr-h1 (JHRE). Based on this mechanism, we developed a system that can be used to screen compounds for JH agonist and antagonist.

［Keywords］

juvenile hormone, Kru¨ppel homolog 1, methoprene tolerant, steroid receptor coactivator, JH response element, JH screening system

［Background］

Juvenile hormone (JH) is an insect-specific hormone that regulates various aspects of development and physiology in insects including molting, metamorphosis and reproduction. It has attracted attention as a target for developing highly selective insecticide due to variations in the chemical structures among many insect groups. However, the mode of action of JH is largely unknown. In this study, we analyzed the transcriptional regulatory mechanism of Kr-h1, a repressor of insect metamorphosis, to clarify the molecular mode of action of JH and develop a screening system for JH agonist and JH antagonist.

［Results and Discussion］

1. We cloned Kr-h1 from B. mori and revealed that Krh1 was induced by JH in individuals and a cell line of B. mori. Furthermore, we cloned two JH receptor genes, Met and its partner gene, steroid receptor coactivator (SRC).
2. By reporter assays using the B. mori cell line, we identified a DNA sequence (JH response element, JHRE) that is indispensable for the response to JH, from the upstream region of Kr-h1 gene (Fig. 1).
3. Analysis using a human cell line clearly demonstrated that upon acceptance of JH, Met forms a complex with SRC, and the JH/Met/SRC complex activates Kr-h1 by interacting with JHRE (Fig. 2).
4. JHRE, Met and SRC are conserved in other insect species with available genome information such as fruit fly, honeybee, red flour beetle and aphid. Therefore the mechanism of Kr-h1 induction by JH via the complex of JHRE and Met/SRC protein complex seems to be common in insects.
5. Utilizing a B. mori cell line and JHRE, we developed an efficient system for screening JH agonists and antagonists (Fig. 3). This screening system enables us to search novel IGR against lepidopteran insect pests.

1. The JHRE, Met and SRC genes will be cloned from important insect pests such as brown planthopper to establish an efficient system for screening JH agonists and antagonists which are specific to target insects. This may lead to the development of highly-selective insecticides.

Fig.1. The JH response element of Kr-h1 (JHRE, 141bp) is located in ~2 kb upstream from the Kr-h1 transcription start site, and contains a canonical E-box sequence (CACGTG) for binding of the bHLH transcription factor.

Fig.2. Schematic representation of the mechanism of JHmediated induction of Kr-h1. �@ Met accepts JH as a ligand, �A JH-liganded Met forms a complex with SRC, �B JH/BmMet2/ BmSRC complex binds to JHRE of Kr-h1, �C Kr-h1 is activated, �D Kr-h1 represses larval- pupal metamorphosis

Fig.3. Screening system using a B. mori cell line and JHRE. Reporter assay using a B. mori cell line introduced with JHRE-luciferase reporter plasmid enables us to effectively search JH agonists (left) and antagonists (right) by measuring reporter activity after application of candidate compounds only or candidate compounds with JH, respectively.

 

[Reference]

1. Kayukawa T, Minakuchi C, Namiki T, Togawa T, Yoshiyama M, Kamimura M, Mita K, Imanishi S, Kiuchi M, Ishikawa Y, Shinoda T (2012) Transcriptional regulation of juvenile hormone-mediated induction of Kru¨ppel homolog 1, a repressor of insect metamorphosis Proceedings of the National Academy of Sciences USA 109(29): 11729-11734