Binding of Escherichia coli heat-stable enterotoxin (STa) to its intestinal receptor is critical to the initiation of toxin-induced secretion and diarrheal disease. However, the action of STa may be a result of molecular mimicry; the primary role for the STa receptor may be to mediate the action of one or more members of the guanylin family of mammalian peptide ligands. The goal of this application is to elucidate the in vivo function of guanylin. This application will address three specific aims: 1) We will test the hypothesis that cis-active elements of the guanylin gene confer cell-specific expression to intestinal epithelial cells. We will use in vitro transcriptional assays with guanylin-luciferase reporter gene constructs and in vivo transgenic mice with guanylin-human growth hormone constructs to study elements which regulate guanylin gene expression. Identification of guanylin promoter regions that direct expression to villus enterocytes and goblet cells may enhance strategies for expression of guanylin in the intestine in proximity to mucin, the guanylin receptor and the cystic fibrosis transmembrane regulator (CFTR). 2) We will make transgenic mice with intestinal specific promoter-guanylin gene constructs to test the hypothesis that intestinal overexpression of guanylin will result in increased basal intestinal Cl secretion. We will determine the effect of overexpression of guanylin on basal and guanylin/STa stimulated intestinal secretion, and guanylin-STa receptor (G-STaR) expression (G- STaR mRNA, guanylate cyclase activity and ligand binding). Transgenic lines will not only be useful for characterization of the in vivo effects of guanylin but they may also be useful as a model of secretory diarrhea or in treating the meconium ileus equivalent seen in the partially corrected CF mouse model. 3) We will use the technique of targeted disruption of the guanylin gene, to test the hypothesis that loss of guanylin expression in the intestine will decrease net intestinal fluid secretion in vivo. We will characterize intestinal secretion in this loss of function model akin to the studies described in Aim 2 for the complementary overexpression model. This """"""""knock-out"""""""" animal model may also be useful to study the actions of other guanylin-related pesticides, e.g., uroguanylin or aberrant synthetic guanylins and for future studies involving crossbreeding to animals in which the G-STaR gene has been targeted. In summary, we will use molecular genetic approaches to define the in vivo role and cellular localization of guanylin. We will develop animal models which be useful to address the specific aims of this proposal and to elucidate the mechanisms of diarrheal disease as well as basal intestinal Cl secretion mediated through the CFTR.
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