Intestinal permeability must be precisely regulated to allow for paracellular uptake of water and nutrients while providing a barrier between the lumen and interstitium. We and others have demonstrated physiological regulation of intestinal permeability as a consequence of Na+-glucose co-transport. Intestinal permeability is also altered by infection with pathogenic bacteria and in idiopathic inflammatory bowel disease. As the rate-limiting step in the paracellular pathway, the tight function is the regulator of intestinal permeability. We have used a reductionist system of Na+-glucose co-transport-dependent tight junction regulation to demonstrate that phosphorylation of the regulatory light chain of myosin II is a target of the signalling cascade activated by Na+-glucose co-transport. This phosphorylation occurs at Ser-19, a site which is known to activate actomyosin contraction. Thus, contraction of the perijunctional actomyosin ring has been proposed as a mechanism of cytoskeletal tight junction regulation. This proposal will test the hypothesis that myosin light chain phosphorylation leads to specific tight junction modifications which form the basis for physiologic regulation of paracellular permeability. In order to dissect the events triggered by myosin light chain phosphorylation from those activated by Na+-glucose co-transport, we will express a truncated continuously active myosin light chain kinase (tMLCK) in a human intestinal epithelial cell line (Caco-2) under the control of an inducible promoter. This system will be used to evaluate functional, structural, and biochemical tight junction alterations that occur as a consequence of myosin light chain phosphorylation. Alterations in tight junction permeability after myosin light chain phosphorylation will be correlated with structural alterations using confocal immunofluorescent localization of tight junction-associated proteins and electron microscopy. Altered distribution, association, and phosphorylation of tight junction-associated proteins will also be evaluated as a function of changes in myosin light chain phosphorylation. The significance of these studies is that they are expected to add to our understanding of the molecular mechanisms of tight junction regulation. An ultimate goal is that the results of these and other studies will lead to understanding of the molecular mechanisms of deranged intestinal permeability in human disease and allows for novel therapeutic interventions.
