Enhanced permeability of intestinal epithelium is a key mechanism of inflammatory diseases of the gut. Such intestinal leakiness exposes internal organs to luminal microbial products, therefore exaggerating mucosal inflammation and increasing the risk of systemic inflammatory responses. Breakdown of the intestinal barrier is caused by disassembly of specialized epithelial structures, tight junctions (TJs) and adherens junctions (AJs). Many inflammatory mediators, including cytokines, nitric oxide and reactive oxygen species, are known to disrupt AJ and TJ structure. Understanding mechanisms of epithelial junctional disassembly during intestinal inflammation represents the major goal of the proposed study. An emerging view is that TJs and AJs undergo a continuous remodeling consisting of the removal of aging junctional components from the plasma membrane by endocytosis and delivery of new TJ/AJ proteins via exocytosis. A central innovative hypothesis of this proposal implies that intestinal inflammation interrupts a steady-state remodeling of epithelial AJs and TJs by blocking vesicle-mediated exocytosis of junctional proteins. This suppression of AJ/TJ exocytosis occurs via inhibition of expression and/or activity of proteins regulating vesicle fusion with the plasma membrane, namely the N-ethylmaleimide sensitive factor (NSF), soluble NSF receptors (SNAREs) and NSF- attachment protein (1SNAP). Dysfunction of SNARE/NSF/1SNAP-mediated trafficking of junctional proteins is likely to eventuate in the defective AJ/TJ structure and increased intestinal barrier permeability. We will test this hypothesis in the following Aims: (1): to investigate the involvement of SNARE-mediated exocytosis in regulation of epithelial junctional structure and functions in vitro and in vivo;(2) to determine the role of oxidative modification of NSF in free-radical induced disassembly of epithelial junctions;3) to analyze the role of 1SNAP in disassembly and recovery of epithelial junctions during mucosal damage and restitution.
These aims will be accomplished using in vitro intestinal epithelial cell monolayers exposed to proinflammatory mediators as well as in vivo murine models of intestinal inflammation. Vesicle fusion machinery will be analyzed by a combination of biochemical (biotinylation, immunoprecipitation, affinity chromatography), immunocytochemical and genetic (siRNA knock-down of SNARE proteins, overexpression of NSF and 1SNAP mutants) approaches. Significance: the proposed study will provide new insights into fundamental mechanisms of intestinal mucosal injury during inflammation. Understanding these mechanisms will potentially provide new therapeutic targets to prevent breakdown of the intestinal barrier in patients with digestive diseases.

Public Health Relevance

The proposed research is aimed to understand mechanisms underlying disruption and restoration of the intestinal epithelial barrier. The barrier breakdown is a common manifestation of different gastroenterological disorders including ulcerative colitis, Chron's disease, celiac diseases and infectious colitis. Furthermore, dysfunctions of the gut barrier contribute to the development of other diseases such as septic shock, alcoholic liver disease and type I diabetes. This project will provide new insights into understanding the pathogenesis of gastrointestinal disorders by exploring a novel epithelium-related mechanism involving in initiation and/or exaggeration of mucosal inflammation. Furthermore, it may provide novel targets for pharmacological prevention of the intestinal barrier breakdown and for accelerated healing of the injured gut mucosa. This may result in decreased morbidity and mortality of a large cohort of patients with inflammatory disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK083968-05
Application #
8662754
Study Section
Gastrointestinal Mucosal Pathobiology Study Section (GMPB)
Program Officer
Carrington, Jill L
Project Start
2010-04-01
Project End
2015-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
5
Fiscal Year
2014
Total Cost
$307,110
Indirect Cost
$101,685
Name
Virginia Commonwealth University
Department
Genetics
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
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