This application for continued support will focus on studying how protein-protein interactions among the cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance protein 4 (MRP4) and inducible nitric oxide synthase (iNOS) contribute to the diarrheal symptom observed in Ulcerative Colitis (UC). Our unifying hypothesis is that CFTR, NHERF2, MRP4 and iNOS form a macromolecular complex at or near the plasma membrane in gut epithelial in UC and this macromolecular complex is required for the pathogenic process of diarrhea observed in UC. Specifically, we proposed that iNOS is overexpressed at the plasma membrane of gut epithelia in UC, resulting in the production of sustained high level of nitric oxide (NO) which triggers NO-cGMP pathway and generates compartmentalized cGMP. Since iNOS forms a macromolecular complex with CFTR, NHERF2 and MRP4 at or near the plasma membrane, this NO-dependent compartmentalized cGMP triggers the hyperactivation of CFTR chloride channels and thus causes diarrhea phenotype. Disruption of the macromolecular complex will abolish the functional coupling of iNOS-dependent cGMP generation and CFTR Cl- channel function.
The specific aims of this proposal are:
Specific Aim 1. To test the hypothesis that iNOS is overexpressed in UC-affected human gut epithelia and to test whether it is coupled to CFTR and MRP4 and upregulates CFTR Cl- channel function Specific Aim 2. To test the hypothesis that iNOS, CFTR, and MRP4 form a macromolecular complex at or near the plasma membrane of gut epithelial cells and that this macromolecular complex is required for the pathogenic process of diarrhea observed in UC. The proposed studies will not only help us better understand the mechanisms underlying the diarrhea symptom commonly observed in UC, but probably provide novel targets and methods for therapeutic interventions of the disease. Therefore, the studies proposed in this application will have clinical relevance for many individuals suffering from UC and also for those suffering from certain forms of secretory diarrheas.

Public Health Relevance

In this grant proposal, we will address a novel mechanism of how protein-protein interactions can contribute to the progression of ulcerative colitis (UC)-related diarrhea. We hypothesize that the macromolecular complex of iNOS (the inducible Nitric Oxide Synthase), CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), NHERF2 (Na+/H+ exchanger regulatory Factor 2) and MRP4 (Multiple drug related protein 4) is spatially and temporally coupled to cGMP (cyclic guanosine monophosphate) generation which causes CFTR-dependent chloride secretion and thus diarrhea in UC. By targeting this macromolecular complex, we may find ways to control fluid secretion and thereby control or cure the disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK080834-05A1
Application #
8505781
Study Section
Clinical, Integrative and Molecular Gastroenterology Study Section (CIMG)
Program Officer
Grey, Michael J
Project Start
2008-04-01
Project End
2013-07-31
Budget Start
2013-04-01
Budget End
2013-07-31
Support Year
5
Fiscal Year
2013
Total Cost
$117,864
Indirect Cost
$39,288
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
Zhang, W; Zhang, X; Zhang, Y H et al. (2016) Lumacaftor/ivacaftor combination for cystic fibrosis patients homozygous for Phe508del-CFTR. Drugs Today (Barc) 52:229-37
Hildebrandt, Ellen; Ding, Haitao; Mulky, Alok et al. (2015) A stable human-cell system overexpressing cystic fibrosis transmembrane conductance regulator recombinant protein at the cell surface. Mol Biotechnol 57:391-405
Balogh, Andrea; Shimizu, Yoshibumi; Lee, Sue Chin et al. (2015) The autotaxin-LPA2 GPCR axis is modulated by γ-irradiation and facilitates DNA damage repair. Cell Signal 27:1751-62
Moon, Changsuk; Zhang, Weiqiang; Ren, Aixia et al. (2015) Compartmentalized accumulation of cAMP near complexes of multidrug resistance protein 4 (MRP4) and cystic fibrosis transmembrane conductance regulator (CFTR) contributes to drug-induced diarrhea. J Biol Chem 290:11246-57
Lin, Sui; Ikegami, Machiko; Moon, Changsuk et al. (2015) Lysophosphatidylcholine Acyltransferase 1 (LPCAT1) Specifically Interacts with Phospholipid Transfer Protein StarD10 to Facilitate Surfactant Phospholipid Trafficking in Alveolar Type II Cells. J Biol Chem 290:18559-74
Riazanski, Vladimir; Gabdoulkhakova, Aida G; Boynton, Lin S et al. (2015) TRPC6 channel translocation into phagosomal membrane augments phagosomal function. Proc Natl Acad Sci U S A 112:E6486-95
Arora, Kavisha; Sinha, Chandrima; Zhang, Weiqiang et al. (2015) Altered cGMP dynamics at the plasma membrane contribute to diarrhea in ulcerative colitis. Am J Pathol 185:2790-804
Sinha, Chandrima; Zhang, Weiqiang; Moon, Chang Suk et al. (2015) Capturing the Direct Binding of CFTR Correctors to CFTR by Using Click Chemistry. Chembiochem 16:2017-22
Moon, Changsuk; Zhang, Weiqiang; Sundaram, Nambirajan et al. (2015) Drug-induced secretory diarrhea: A role for CFTR. Pharmacol Res 102:107-12
Sinha, Chandrima; Ren, Aixia; Arora, Kavisha et al. (2015) PKA and actin play critical roles as downstream effectors in MRP4-mediated regulation of fibroblast migration. Cell Signal 27:1345-55

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