20-Hydroxyecdysone Suppression of Juvenile Hormone Response

摘要：表皮甾体激素（Ecdysteroids， JH）和幼体激素（juvenile hormone， JH）是调节昆虫生命周期中各种发育事件的主要激素。蜕皮激素的多种生物学效应是由蜕皮激素受体（EcR）及其异二聚体伴侣超囊泡（USP）介导的。JH作用的分子机制尚不清楚。JH和表皮甾体激素的生物学效应表明这两种激素之间存在交叉作用。在JH-和20-羟基蜕皮激素（20E）响应的JH酯酶（jhe）基因启动子区发现JH应答元件（JHRE）和蜕皮激素受体（EcR）缺陷的果蝇黑ogaster细胞系（L57）建立了一个强大的系统。在L57细胞中，JHRE控制下的一个报告基因被JH诱导，JH诱导的表达被20E以剂量和时间依赖性的方式抑制。从L57细胞中分离的核蛋白与JHRE特异性结合，用20E预处理核蛋白可阻止这种结合。这种20E抑制jh诱导的基因表达是通过EcR介导的。初步研究表明，EcR不直接与含有JHRE的启动子结合。20E抑制JH的作用需要与EcR的配体结合，而不需要与DNA结合。这些结果表明，EcR通过一种“非经典”的作用方式抑制jh诱导的基因表达，即不直接结合蜕皮激素反应元件。这是提出EcR非经典作用的系统的第一个例子。该系统将用于研究EcR的非经典作用及其与JH信号转导级联成分的串扰机制。本提案的两个具体目标是：(1)研究20E抑制JH应答的机制；(2)鉴定和表征20E和JH之间串扰的重要蛋白。在D. melanogaster EcR的配体结合域中选择氨基酸的突变将用于验证EcR的非经典作用。利用RNA干扰（RNAi）介导的usp、果蝇激素受体38、78和Seven-up基因的沉默，研究它们在20E抑制JH应答中的作用。酵母双杂交试验将用于鉴定与EcR相互作用的蛋白质。ecr相互作用蛋白将通过(i) rnai介导的L57细胞中表达的沉默，（ii）拉下和双杂交试验，（iii）研究D. melanogaster组织中RNA的表达，以及（iv）分析D. melanogaster突变体中编码所鉴定蛋白质的基因的JH和20E反应来表征。

Title: 20-hydroxyecdysone suppression of juvenile hormone actionPI: Subba Reddy PalliAbstract:Ecdysteroids and juvenile hormone (JH) are major hormones that regulate various developmental events during an insect's life cycle. The diverse biological effects of ecdysteroids are mediated by ecdysone receptor (EcR) and its heterodimeric partner, ultraspiracle (USP). The molecular mechanisms of JH action are not well understood. The biological effects of JH and ecdysteroids suggest a cross talk between these two hormones. JH response elements (JHRE) identified in the promoter region of JH- and 20-hydroxyecdysone (20E)-responsive JH esterase (jhe) gene and ecdysone receptor (EcR) deficient Drosophila melanogaster cell line (L57) were used to develop a robust system. In L57 cells, a reporter gene placed under the control of JHRE is induced by JH, and the JH-induced expression is suppressed by 20E in a dose- and time-dependent manner. The nuclear proteins isolated from L57 cells specifically bound to JHRE and this binding was prevented by pretreatment of nuclear proteins with 20E. This 20E suppression of JH-induced gene expression is mediated through EcR. Preliminary studies showed that EcR does not directly bind to JHRE containing promoter. Ligand binding but not DNA binding of EcR is required for 20E suppression of JH action. These results suggest that the EcR suppresses JH-induced gene expression though a "nonclassical" mode of action, i.e., without binding to ecdysone response elements directly. This is the first example of a system where a nonclassical action for EcR is suggested. This system will be used to study the mechanisms involved in the nonclassical action of EcR and its cross talk with the components of JH signal transduction cascade. The two specific objectives of this proposal are (1) to study the mechanism for 20E suppression of JH response and (2) to identify and characterize proteins that are important in cross talk between 20E and JH. Mutations to select amino acids in the ligand binding domain of D. melanogaster EcR will be used to verify nonclassical action of EcR. RNA interference (RNAi)-mediated silencing of usp, Drosophila hormone receptors 38 and 78, and Seven-up genes will be employed to study their role in 20E suppression of JH response. A yeast two-hybrid assay will be used to identify proteins that interact with EcR. The EcR-interacting proteins will be characterized through (i) RNAi-mediated silencing of their expression in L57 cells, (ii) pull down and two-hybrid assays, (iii) studies on expression of RNA in D. melanogaster tissues and (iv) analysis of JH and 20E response in D. melanogaster mutants for genes coding for the identified proteins.