Physiological Regulation of Intestinal Epithelial Transport and Barrier Function

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Defective intestinal transport is a central component of many intestinal diseases. Such transport, in the form of nutrient, ion, and water absorption, is accomplished by specific transcellular transporters as well as passive paracellular movement across the epithelial tight junction. An emerging view is that individual transporters can regulate one another as well as paracellular permeability, resulting in coordinate regulation of intestinal transport. This concept is supported by our studies of Na+-glucose cotransport-dependent tight junction regulation showing that SGLT1, the apical Na+-glucose cotransporter, activates a signal transduction cascade that sequentially triggers delivery of NHE3, an apical Na+-H+ exchanger, to the plasma membrane, increased apical Na+-H+ exchange, myosin light chain (MLC) kinase activation, MLC phosphorylation, actomyosin contraction, and increased tight junction permeability. Despite these and other advances, the mechanisms by which vesicular transport, protein interactions, and actomyosin contraction effect regulation of intestinal epithelial transport and barrier function remain largely undefined. This critical gap limits our ability to understand the mechanisms of diseases with intestinal transport and barrier dysfunction, including infectious, inflammatory, and malabsorptive diarrheal diseases. Thus, the objectives of this application are to define the mechanisms by which signal transduction pathways that activate membrane traffic and modify protein interactions are involved in regulation of intestinal transport and barrier function. We will accomplish these objectives through three specific aims: 1. To define the role of ezrin in acute regulation of protein delivery to the plasma membrane, 2. To identify the mechanisms that define the dynamic behavior of proteins at the tight junction, and 3. To define the mechanisms and significance of actomyosin-dependent tight junction maintenance and regulation. These studies will be performed using in vitro and in vivo models that include imaging of fluorescent fusion proteins expressed in living cells and tissues and in vivo analysis of immune activation secondary to transgenic manipulation of tight junction permeability. As a result we will significantly advance our understanding of the mechanisms by which membrane, protein, and cytoskeletal dynamics contribute to regulation of transport and barrier function. In addition to providing new fundamental knowledge this is expected to have significant positive effects on human health because it will allow the rational development of new therapeutic strategies for diseases with deficient transport or barrier function. SUMMARY: The intestinal epithelium must balance nutrient transport with the need for a barrier that keeps the intestinal contents separate from the remainder of the body. Recent data have shown that these functions are coordinately regulated, which is consistent with the fact that both are frequently compromised in disease. The proposed studies will advance mechanistic understanding of this coordinate regulation and may therefore lead to the development of novel approaches for treatment of intestinal disease.