Steroid signaling controls a wide array of cell-specific responses, ranging from carbohydrate metabolism and inflammation, to cytoskeletal reorganization and tissue remodeling during development. Steroid hormones bind to nuclear receptor proteins that primarily function as ligand-activated transcription factors. Our current studies are focused on understanding the role of the insect steroid 20-hydroxyecdysone in controlling blood meal digestion in Aedes aegypti mosquitoes, a biological vector for Dengue virus and yellow fever. Moreover, through a collaboration with Dr. Bill Montfort in our department, we are investigating the structure and function of an Ae. aegypti nuclear receptor protein called E75A that is induced by ecdysone signaling in the fat body of blood fed adult female mosquitoes. We use a variety of genomic and proteomic methodologies in combination with molecular genetic and biochemical approaches to achieve our research goals.
Regulation of protease gene expression in the midgut of Ae. aegypti mosquitoes
The primary focus of this project is to identify the molecular mechanism of coordinate expression of protease genes in the mosquito midgut after blood meal feeding. We have taken two complementary approaches to investigate this process. One involves using organ culture system based on dissected midgut tissue, and the other a molecular genetic approach involving RNA interference (RNAi) methods in adult mosquitoes. Using the organ culture system, we have shown that ecdysone addition to midgut cultures induces expression of the late trypsin gene. These novel findings in adult mosquitoes indicate that steroid signals emanating from the fat body (insect liver) play a key role in activating protease gene expression in the midgut to maximize blood meal digestion. We have also begun to investigate the contribution of other fat body factors, as well as components of the blood meal, that may be involved in augmenting ecdysone signaling in the mosquito midgut.
Structure and function of the Ae. aegypti E75A nuclear receptor protein in the fat body
Blood meal feeding induces the synthesis of ecdysone in the ovaries of female mosquitoes, which in turn, binds to and activates the ecdysone receptor protein in multiple tissues. In the fat body, activated ecdysone receptor induces the expression of numerous downstream genes, one of which is the E75A nuclear receptor protein which is a heme-containing transcription factor that regulates the expression of egg protein genes (vitellogenins). Recent studies in Drosophila have suggested that E75A transcriptional regulatory activity may be controlled by nitric oxide binding to the heme group. Since the mosquito blood meal contains large amounts of heme, and feeding induces the synthesis of both ecdysone and nitric oxide, we are testing the hypothesis that the Ae. aegypti E75A protein functions as a metabolic sensor in the fat body.
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Isoe, J., Kunz, S., Manhart, C., Wells, M.A. and Miesfeld, R.L. (2006). Regulated expression of microinjected DNA in adult Aedes aegypti mosquitoes. Insect Mol. Biol., in press.
Wang, J., Cai, Y.. Pen, R., Chauhan, S., Miesfeld, R.L. and Ittmann, M. (2006). Increased Expression of the Metastasis Associated Gene Ehm2 in Prostate Cancer, The Prostate, in press.
Chauhan, S., S. Kunz, K. Davis, J. Roberts, G. Martin, M. Demetriou, T. Sroka, A. Cress, and R. Miesfeld. 2004. Androgen control of cell proliferation and cytoskeletal reorganization in human fibrosarcoma cells: Role of RhoB signaling. Journal of Biological Chemistry 279: 937-944.
Bloom, J.W., J. Chacko, M.S. Lohman, M. Halonen, F.D. Martinez, and R. Miesfeld. 2004. Differential control of eosinophil survival by glucocorticoids. Apoptosis 9: 97-104.
Kunz, S., R. Sandoval, P. Carlsson, J. Carlstedt-Duke, J. Bloom, and R. Miesfeld. 2003. Identification of a novel glucocorticoid receptor mutation in budesonide-resistant human bronchial epithelial cells. Molecular Endcrinology 17: 2566-2582.
Chauhan, S., C. Leach, S. Kunz, J. Way, J. Bloom, and R. Miesfeld. 2003. Glucocorticoid regulation of human eosinophil gene expression. Journal of Steroid Biochemistry and Molecular Biology 84: 441-452.
