Breakdown of the intestinal epithelial barrier represents an early and important manifestation of various gastrointestinal disorders including inflammatory bowel disease. The integrity of the epithelial barrier is regulated by cellular structures known as tight junctions (TJs) and adherens junctions (AJs). Disruption of TJs and AJs underlies increased permeability of inflamed intestinal epithelium, whereas junctional reassembly is crucial for recovery of the epithelial barrier during mucosal restitution. Understanding mechanisms of AJ and TJ remodeling during intestinal epithelial damage and restitution represents the major goal of the proposed study. TJs and AJs associate with the actin cytoskeleton which is a crucial regulator of junctional reorganizations and integrity. Actin filaments are highly dynamic polymers undergoing a constant turnover (depolymerization and polymerization). Such a turnover of filamentous (F) actin is critical for a variety of cellular functions, however its involvement in AJ/TJ regulation remains poorly investigated. A central innovative hypothesis of this proposal implies that F-actin turnover plays a key role in reorganizations of epithelial AJs and TJs during mucosal damage and restitution. Particularly, I propose that accelerated turnover of perijunctional actin filaments mediates both AJ/TJ disassembly and reformation of nascent AJ-like junctions, whereas decreased F-actin turnover is prerequisite for the establishment of TJs and barrier resealing. This hypothesis will be tested in the following Aims: (1) To examine the role of actin filament turnover in disassembly and reformation of epithelial AJs and TJs;(2) to investigate the involvement of actin-depolymerizing factor (ADF)/cofilin proteins in junctional remodeling during intestinal epithelial damage and restitution;3) to analyze the role of F-actin cross-linking proteins in disassembly and reestablishment of epithelial junctions.
These aims will be accomplished using in vitro intestinal epithelial cell monolayers exposed to inflammatory mediators as well as in vivo mouse model of colitis and tissue biopsies of human patients. Turnover of actin filaments and functions of actin- depolymerizing and cross-linking proteins will be analyzed by a combination of biochemical (actin- monomer sequestration, immunoblotting, detergent fractionation), immunocytochemical and genetic (siRNA-mediated knock-downs, expression of dominant-negative or constitutively active mutants, knockout mice) approaches. Significance: the proposed study will provide new insights into fundamental mechanisms of intestinal mucosal injury during inflammation. Understanding these mechanisms will provide new therapeutic targets to prevent breakdown and enhance reparation of the gut 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, Crohn'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 #
7R01DK084953-03
Application #
8475848
Study Section
Gastrointestinal Mucosal Pathobiology Study Section (GMPB)
Program Officer
Carrington, Jill L
Project Start
2010-07-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
3
Fiscal Year
2012
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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Wang, Dongdong; Naydenov, Nayden G; Feygin, Alex et al. (2016) Actin-Depolymerizing Factor and Cofilin-1 Have Unique and Overlapping Functions in Regulating Intestinal Epithelial Junctions and Mucosal Inflammation. Am J Pathol 186:844-58
Miller, Kelly A; Chaand, Mudit; Gregoire, Stacy et al. (2016) Characterization of V. cholerae T3SS-dependent cytotoxicity in cultured intestinal epithelial cells. Cell Microbiol 18:1857-1870
Wang, Dongdong; Chadha, Gibran K; Feygin, Alex et al. (2015) F-actin binding protein, anillin, regulates integrity of intercellular junctions in human epithelial cells. Cell Mol Life Sci 72:3185-200
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Lechuga, Susana; Baranwal, Somesh; Li, Chao et al. (2014) Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells. Mol Biol Cell 25:3133-46
Naydenov, Nayden G; Feygin, Alex; Wang, Lifu et al. (2014) N-ethylmaleimide-sensitive factor attachment protein α (αSNAP) regulates matrix adhesion and integrin processing in human epithelial cells. J Biol Chem 289:2424-39
Kuo, I-Hsin; Carpenter-Mendini, Amanda; Yoshida, Takeshi et al. (2013) Activation of epidermal toll-like receptor 2 enhances tight junction function: implications for atopic dermatitis and skin barrier repair. J Invest Dermatol 133:988-98
Ghatak, Sayak; Reveiller, Marie; Toia, Liana et al. (2013) Bile acid at low pH reduces squamous differentiation and activates EGFR signaling in esophageal squamous cells in 3-D culture. J Gastrointest Surg 17:1723-31

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