Location: Vegetable Crops ResearchTitle: Juvenile Hormone Regulation of Drosophila Epac - A Guanine Nucleotide Exchange Factor for Rap1 Small GTPase) Author
Submitted to: Molecular and Cellular Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/6/2009
Publication Date: 2/21/2009
Citation: Wang, J., Lindholm, J.R., Willis, D.K., Orth, A., Goodman, W.G. 2009. Juvenile Hormone Regulation of Drosophila Epac - A Guanine Nucleotide Exchange Factor for Rap1 Small GTPase. Molecular and Cellular Endocrinology. 305(1-2):30-37. Interpretive Summary: Insect juvenile hormones play important roles in the development of animal and plant insect pests. Several commercial insecticides such as methoprene are chemically synthesized juvenile hormone (JH) derivatives that aid in the control of insect pests in both animals and plants. We used microarrays containing probes for all 14,010 genes of the fruit fly Drosophila melanogaster to identify the array of genes that are affected by addition of juvenile hormone. We detected 32 genes whose expression was either increased or decreased by exposure to juvenile growth hormone. A subset of these genes were further analyzed using real-time RT-PCR and this technique confirmed that 3 out of 3 genes that were thought to increase actually did increase. One of these genes, Epac is significantly induced by juvenile hormone. This paper describes the detailed characterization of the induction of Epac by JH including the time course of induction, effect of JH concentration, and specificity of induction by JH analogs and angonists. The identification of the juvenile hormone regulatory cascade may provide novel targets for future insecticide action of use to both plant and animal agriculture.
Technical Abstract: Previously, we utilized a microchip array encompassing probes for 14,010 genes of Drosophila melanogaster to analyze the effect of (10R) juvenile hormone III (JH) on genome-wide gene expression in Drosophila S2 cells. Treatment with JH yielded a collection of 32 gene transcripts that demonstrated a significant change in abundance with hormone treatment when compared to a control treatment with methyl linoleate which has a similar structure to JH but displays no hormonal activity. In this work, we used real-time quantitative reverse transcription PCR (qRT-PCR) to characterize the effects of the treatment of S2 cells with JH and JH analogs on the expression of Epac (Exchange Protein directly Activated by Cyclic AMP; FBgn0085421), a guanine nucleotide exchange factor for the small GTPase Rap1. The relative expression ration (RER) of Epac displayed a rapid, dose-dependent increase with JH treatment. The minimal level of hormone needed to elicit a response was 100 ng/ml while maximal Epac expression was achieved at 500 ng/ml. Time course studies indicated a significant rise 1 h after treatment, with the peak Epac RER occurring at 4 h. Epac transcriptional activity induced by JH was also verified by real-time qRT-PCR using an intron-specific probe. S2 cells were challenged with various JH homologs, analogs, and metabolites to determine the specificity of the enhanced RER of Epac gene expression. Methoprene, a synthetic agonist, and (10R) JH III displayed the greatest activity while (10S) JH III, (10R,11S) JH I, bisepoxy JH III and methyl farnesoate were less active. MLA and JH III acid, a metabolic product of JH degradation, were inactive. The molting hormone 20-hydroxyecdysone did not increase Epac RERs above control levels. The response of late third instar (96 h) Drosophila to the potent agonist methoprene was tested by exposing the insects to diet containing 500 ng/g diet; significantly higher Epac RERs were noted at 12 and 18 h after exposure. JH had no effect on Epac transcript levels in the human cell line HEK-293.