Intestinal barrier dysfunction contributes to progression of gastrointestinal and systemic disease. Over the previous two cycles of this award we have i) discovered mechanisms by which myosin light chain kinase (MLCK) regulates intestinal epithelial tight junction barrier function, in vitro and in vivo;ii) developed tools to prevent this regulation in cultured monolayers and experimental animals;and iii) demonstrated that increasing or reducing intestinal epithelial MLCK activity can enhance or reduce, respectively, progression of both experimental inflammatory bowel disease (IBD) and graft versus host disease (GVHD). Although the tools developed have been extremely informative, they are not suitable for translation to human patients. This is primarily because it is not possibe to specifically inhibit intestinal epithelial MLCK enzymatic activity without also inhibiting smoot muscle MLCK which results in severe, sometimes fatal, toxicities. Further, MLCK serves important functions other than tight junction regulation in intestinal epithelia, including promotin of wound healing. Thus, there is a fundamental gap that separates our previous elucidation of mechanisms and clinicopathologic significance of barrier regulation in disease from development of strategies that can be used to modulate intestinal epithelial tight junction function for therapeutic purposes. This proposal seeks to bridge that gap by building on our recent observations regarding regulation of the MLCK-myosin phosphatase axis in disease. Specifically, we will focus on understanding trafficking of the MLCK1 splice variant. We have shown that tumor necrosis factor (TNF) or chronic disease cause MLCK1 recruitment to the perijunctional actomyosin ring (PAMR), to regulate tight junction permeability. Moreover, we have developed a small molecule inhibitor that blocks this trafficking and is remarkably effective in experimental IBD. Here we propose to define the molecular mechanisms of basal and TNF-induced MLCK1 trafficking and to characterize the therapeutic potential of newly-discovered trafficking inhibitors in experimental IBD and GVHD. Our preliminary data also demonstrate an unexpected, essential, in vivo role of the myosin phosphatase regulatory subunit MYPT1 in mucosal homeostasis. MYPT1 regulates MLC phosphatase activity and specificity and thereby opposes MLCK function. Thus, understanding the means by which MYPT1 loss becomes catastrophic is expected to provide additional new insights into the functions of the MLCK-myosin phosphatase axis in homeostasis and disease. The proposal is innovative because it will define novel regulatory mechanisms and will result in a major shift in our understanding of means to correct barrier function and actomyosin contractile status for therapeutic benefit. The proposed research is significant because it will link specific mechanisms of barrier loss to disease and identify novel therapeutic approaches. Finally, in addition to benefitting diseases associated with intestinal barrier loss, the concepts and tools developed will be applicable to barrier restorative therapy for diseases of other organs that are driven by epithelial or endothelial barrier dysfunction.
The proposed research is relevant to public health because discovery of the mechanisms that regulate tissue barriers that separate sterile internal compartments from those colonized by microbiota, e.g. the intestinal lumen, and agents that modify this these processes will provide a foundation for development therapeutic interventions to maintain or restore barrier function in disease. This work will therefore directly support the overall NIH mission of developing fundamental knowledge that will help reduce the burden of human disease and promote the NIDDK goal of improving digestive health.
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|Lingaraju, Amulya; Long, Tiha M; Wang, Yitang et al. (2015) Conceptual barriers to understanding physical barriers. Semin Cell Dev Biol 42:13-21|
|Edelblum, Karen L; Singh, Gurminder; Odenwald, Matthew A et al. (2015) Î³Î´ Intraepithelial Lymphocyte Migration Limits Transepithelial Pathogen Invasion and Systemic Disease in Mice. Gastroenterology 148:1417-26|
|Weber, Christopher R; Liang, Guo Hua; Wang, Yitang et al. (2015) Claudin-2-dependent paracellular channels are dynamically gated. Elife 4:e09906|
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