Elucidating the Molecular Modes of Action of Insect Juvenile Hormone.

摘要

Juvenile hormone (JH) controls insect growth, development and reproduction. Despite our understanding of the physiological actions, the molecular modes of action of JH have not been fully elucidated. Several genes important for physiological responses to JH have been determined, but a complete signaling pathway has yet to be described. To identify key components of JH signaling pathways, in vivo and in vitro studies were conducted on Drosophila melanogaster that: identified physiologically relevant gene expression regulated by JH alone, characterized proteins interacting directly with JH, and elucidated genes necessary for JH signaling.;First, genes regulated by JH were identified by microarray analysis in Drosophila S2 cells. 32 genes demonstrated significant ≥ 2-fold change with JH III treatment. JH/analogs treatment led to rapid, dose-dependent increase in a gene of interest: exchange protein directly activated by cyclic AMP (Epac). Late third instar (96 h) Drosophila exposed to diet containing JH analog, methoprene, for 12 or 18 hours showed significant > 1.5 fold increase in relative expression ratio of Epac. An Epac target, RAP1, regulates Drosophila morphogenesis in vivo providing evidence linking molecular actions of JH to insect development.;Second, proteins interacting directly with JH or methoprene were affinity-purified from Drosophila S2 cell lysates and identified by mass spectrometry. Over 150 proteins specifically bound the affinity columns. This study confirmed the known interaction with heat shock protein 83 (Hsp83) and identified novel co-chaperones in the heat shock protein 70 family.;Third, genes necessary for a JH-response through the conserved effector, Kruppel homolog 1 (Kr-h1) were determined by knockdown. A genome-wide Drosophila Kc167 cell-based screening using RNA interference (RNAi) with a luciferase reporter plasmid was performed to identify genes necessary for this JH signaling. The double-stranded RNA (dsRNA)-mediated knockdown of 72 genes strongly affected JH signaling consisting of 57 positive and 15 negative regulators. There were 17 genes that had some effect on JH signaling in these screens that were also upregulated in the microarray by JH treatment. Additionally, 53 genes that were JH/analog affinity-purified also affected JH signaling in these screens. Novel key players in JH signaling identified include E74, E75, Hr4 and Hr38.