Steroid-Induced Gene Expression: A link to Ca[2+]-Regulated Exocytosis

Northwestern University, Evanston IL

Investigators

Abstract

Steroid hormones are small lipophilic regulatory molecules that profoundly influence basic processes of growth, homeostasis, and disease progression in developing organisms. Unlike peptide hormone signals, most of the physiological responses mediated by steroids occur though a nuclear action of the ligand/receptor complex on responding genes (a genomic mechanism). Although much has been learned over the past decade concerning the molecular details of receptor activation and transcriptional regulation of steroid-responsive DNA targets, little is known about how the qualitative differences in gene expression lead to long-term physiological changes in the responding tissue. This remains a fundamental question of cell biology. The fruitfly, Drosophila melanogaster, has contributed much to our understanding of the conserved processes of developmental signaling. It is also an attractive system in which to study steroid responses because only one steroid, 20-hydroxyecdysone (ecdysone), is known to regulate the important postembryonic developmental transitions of molting and metamorphosis. Furthermore, the salivary gland of the third larval stage offers a unique opportunity to correlate changes in gene expression with a tissue-specific physiological response. This is possible because the chromosomes within this tissue are polytenized. Thus, hormone targets are easy to identify because they often form puffs on the chromosomes (areas of local chromatin decondensation) in response to ecdysone. In addition, it is known that the salivary gland will respond to hormone by secreting massive amounts of glue proteins, which are later used by the animal to cement itself to a solid surface during metamorphosis. Because glue secretion and chromosome puffing occur over the same time interval, it has been suggested that one or more products of the puffing cascade control the secretion response. Work conducted in Dr. Andres' laboratory has confirmed the above hypothesis. By investigating the function of the puff genes in a secretion assay in which a GFP-tagged glue protein is used, they have identified a role for E63-1. This protein is directly induced by ecdysone, regulated by calcium ions, and associated with an unconventional myosin that is required for glue secretion. The project supported by this award will test the hypothesis that, as part of the genomic response of the salivary gland to ecdysone, E63-1 is induced and cytoplasmic calcium levels are elevated, and that once E63-1 is complexed with calcium, it acts as a bona fide light chain for myosin VI to influence the movement of secretory granules. The aim of the work is to define the biochemical interaction between the E63-1 and myosin VI proteins, and to identify key molecules important in the ecdysone regulation of calcium levels using genetic screens. The project has the potential to make important contributions to a basic, but not well understood, mechanism of cell biology involving the integration of steroid and calcium signaling pathways. In addition, since most of the personnel conducting this work will be students, the project offers them a unique learning and training experience using a multi- disciplinary approach employing genetic, molecular, biochemical, and pharmacological methodologies