Oxidative stress-induced intestinal epithelial barrier dysfunction has been implicated in the pathogenesis of a wide spectrum of intestinal inflammatory diseases including necrotizing enterocolitis, inflammatory bowel disease and infectious enterocolitis. A significant body of evidence indicates that barrier function of GI tract is disrupted in various GI diseases. Our studies during the past several years have demonstrated that oxidative stress disrupts intestinal epithelial tight junction by phosphorylation of occludin on tyrosine residues and dephosphorylation on threonine residues;these processes involve the intracellular signaling molecules such as c-Src, FAK, PI3K, PP2A, PKC7, PKC6 and PP1. Our studies also demonstrated that EGF, a GI mucosal protective factor, prevents the oxidative stress-induced disruption of tight junctions;this process involved signaling molecules, PLC3, PKC2I, PKC5 and MAP kinases. Our preliminary data have identified two tyrosine residues and two threonine residues in occludin that lie in a highly conserved motif of occludin and suggested that phosphorylation of these residues regulates the integrity of tight junctions. Therefore, we plan to further continue our effort to understand the regulation of phosphorylation of these amino acids in the oxidative stress- induced disruption of intestinal epithelial barrier function and EGF-mediated prevention of oxidative stress effects. On the basis of our results it is hypothesized that: a) c-Src-mediated phosphorylation of occludin on Y398 and Y402 mediates the H2O2-induced destabilization of intestinal epithelial tight junctions, b) H2O2- induced disruption of tight junctions involves of dephosphorylation of occludin on T403 and T404 by down regulation of PKC7, and c) EGF and probiotics prevent oxidative stress-induced tight junction disruption by a MAP kinase-dependent mechanism. We propose to determine that: 1) c-Src directly interacts with occludin and phosphorylates Y398/Y402 in H2O2-treated cells. 2) H2O2-induced Tyr-phosphorylation of claudin-3 plays a role in the disruption of tight junctions. 3) c-Src plays a role in H2O2-induced attenuation of steady state dynamics of tight junction proteins. 4) H2O2 accelerates cell spreading and migration by c-Src-mediated phosphorylation of occludin and claudin-3. 5) PKC6 regulates tight junction integrity by Ser/Thr- phosphorylation of tight junction proteins. 6) Oxidative stress disrupts tight junctions by down regulation of PKC7. 7) Phosphorylation of tight junction proteins on specific Ser/Thr residues regulates their self-association and actin binding affinity. 8) Probiotics protect tight junctions from H2O2 by inducing EGF receptor transactivation. 9) EGF and probiotics ameliorate ischemia/reperfusion-induced tight junction disruption in mouse intestine. 10) EGF and probiotics attenuate the H2O2-induced PKC7 down regulation, alteration of occludin phosphorylation and tight junction disruption in human colonic biopsies. The outcome of these studies has a direct relevance to our understanding of the pathogenesis of many GI diseases and has the potential to contribute to the future development of new therapeutic strategies.

Public Health Relevance

Intestinal diseases such as inflammatory bowel disease and infectious enterocolitis are associated with tissue damage caused by oxidative stress and intestinal epithelial barrier dysfunction. On the basis of our research during the past several years we hypothesized that oxidative stress disrupts intestinal epithelial barrier function by inducing phosphorylation of tight junction proteins, and the protective growth factors such as epidermal growth factor prevent such cellular damage by oxidative stress. We propose to conduct studies to uncover the cellular and molecular mechanisms involved in these processes;and the outcome of these studies are expected to provide knowledge to develop new therapies in the treatment of different intestinal diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK055532-14
Application #
8310058
Study Section
Clinical, Integrative and Molecular Gastroenterology Study Section (CIMG)
Program Officer
Carrington, Jill L
Project Start
1998-09-15
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
14
Fiscal Year
2012
Total Cost
$304,029
Indirect Cost
$98,604
Name
University of Tennessee Health Science Center
Department
Physiology
Type
Schools of Medicine
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
Chaudhry, Kamaljit K; Shukla, Pradeep K; Mir, Hina et al. (2016) Glutamine supplementation attenuates ethanol-induced disruption of apical junctional complexes in colonic epithelium and ameliorates gut barrier dysfunction and fatty liver in mice. J Nutr Biochem 27:16-26
Shukla, Pradeep K; Chaudhry, Kamaljit K; Mir, Hina et al. (2016) Chronic ethanol feeding promotes azoxymethane and dextran sulfate sodium-induced colonic tumorigenesis potentially by enhancing mucosal inflammation. BMC Cancer 16:189
Mir, Hina; Meena, Avtar S; Chaudhry, Kamaljit K et al. (2016) Occludin deficiency promotes ethanol-induced disruption of colonic epithelial junctions, gut barrier dysfunction and liver damage in mice. Biochim Biophys Acta 1860:765-74
Shukla, Pradeep K; Gangwar, Ruchika; Manda, Bhargavi et al. (2016) Rapid disruption of intestinal epithelial tight junction and barrier dysfunction by ionizing radiation in mouse colon in vivo: protection by N-acetyl-l-cysteine. Am J Physiol Gastrointest Liver Physiol 310:G705-15
Samak, Geetha; Chaudhry, Kamaljit K; Gangwar, Ruchika et al. (2015) Calcium/Ask1/MKK7/JNK2/c-Src signalling cascade mediates disruption of intestinal epithelial tight junctions by dextran sulfate sodium. Biochem J 465:503-15
Chaudhry, Kamaljit K; Samak, Geetha; Shukla, Pradeep K et al. (2015) ALDH2 Deficiency Promotes Ethanol-Induced Gut Barrier Dysfunction and Fatty Liver in Mice. Alcohol Clin Exp Res 39:1465-75
Samak, Geetha; Gangwar, Ruchika; Crosby, Lynn M et al. (2014) Cyclic stretch disrupts apical junctional complexes in Caco-2 cell monolayers by a JNK-2-, c-Src-, and MLCK-dependent mechanism. Am J Physiol Gastrointest Liver Physiol 306:G947-58
Rao, R K; Samak, G (2013) Protection and Restitution of Gut Barrier by Probiotics: Nutritional and Clinical Implications. Curr Nutr Food Sci 9:99-107
Dunagan, Mitzi; Chaudhry, Kamaljit; Samak, Geetha et al. (2012) Acetaldehyde disrupts tight junctions in Caco-2 cell monolayers by a protein phosphatase 2A-dependent mechanism. Am J Physiol Gastrointest Liver Physiol 303:G1356-64
Rao, RadhaKrishna; Samak, Geetha (2012) Role of Glutamine in Protection of Intestinal Epithelial Tight Junctions. J Epithel Biol Pharmacol 5:47-54

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