Inflammatory bowel disease (IBD) currently affects 1.4 million Americans. Reduced barrier function has been associated with active IBD and, interestingly, is also present in a subset of healthy, first-degree relatives of Crohn's disease patients. In patients with IBD, reduced barrier function during clinical remission is also associated with increased risk of relapse. Tumor necrosis factor (TNF), a central inflammatory cytokine in IBD, causes barrier dysfunction via myosin light chain kinase (MLCK) activation and subsequent tight junction dysregulation. Moreover, MLCK expression and activity are also increased in IBD patients. These data suggest that TNF- and MLCK-dependent barrier dysfunction contribute to IBD pathogenesis. One MLCK splice variant, MLCK1, contains a unique immunoglobulin-cell adhesion molecule (IgCAM) domain, IgCAM3, that is not present in the other variants of MLCK1. Consistent with a unique role for IgCAM3 in trafficking, MLCK1, but not MLCK2, is concentrated at the perijunctional actomyosin ring and our preliminary data show that MLCK1 trafficking is required for TNF-induced barrier loss in vitro and in vivo. These preliminary data suggest that MLCK1 trafficking is essential to TNF-induced barrier loss in IBD. The aims of this proposal are to i) identify the domains of MLCK1 that are required for MLCK1 trafficking to the perijunctional actomyosin ring, ii) define the role of the chaperone FKBP8 in MLCK1 localization and iii) characterize small molecules that specifically disrupt MLCK1 trafficking to the perijunctional actomyosin ring and reduce increased intestinal permeability in vivo. Although most current therapies directed at IBD are centered on suppressing the immune response (e.g. corticosteroids and anti-TNF therapies), long-term use of these therapies carries significant risk of other complications. Thus, alternative therapies that can be used to maintain remission are needed. We have hypothesized that inhibition of MLCK1 trafficking, which should have very few side effects, will blunt barrier dysregulation induced by TNF and related cytokines and thus help to maintain remission in patients and perhaps prevent IBD development in at-risk individuals. These studies are significant because they will be the first step toward understanding the mechanisms by which MLCK1 trafficking is in involved in TNF-induced barrier dysregulation, and they will define a signaling pathway that can be targeted therapeutically. PUBLIC HEALTH RELEVANCE: Inflammatory bowel disease (IBD), characterized by increased intestinal permeability and reduced barrier function, currently affects 1.4 million Americans. The proposed studies will delineate a novel signaling pathway involved in barrier regulation and will define the characteristics of small molecule drugs that can be used to target that pathway therapeutically. The data generated from these studies will have a significant public health impact, as the development of novel drugs with low toxicity is critical for reducing both the incidence of relapse in IBD and the health risks associated with long-term use of the current immunosuppressive drugs that are currently used to treat IBD.