Mo2042 Identification of a Novel CFTR Interactome in Contributing Diarrhea in Inflammatory Bowel Disease

Abstract

Background: Inflammatory bowel disease (IBD) is a group of disorders in which the gut suffers from inflammation. Among the few predisposing genetic factors identified in IBD, a cGMP/cAMP-regulated chloride (Cl-) channel CFTR has emerged as one. Our lab and others have reported involvement of CFTR in formation of multiple protein complexes that can regulate the channel function in healthy and diseased states. In our current study, we aim to elucidate a molecular mechanism in the form of a signaling protein complex of CFTR that contributes to disease progression in IBD. Method: (a) Protein-protein interactions were studied using biochemical methods (protein pairwise binding assay,macromolecular complex assembly assay, and co-immunoprecipitation) and In Vitro methods for studying protein interaction in live cell (N-FRETc) in HEK293 cells. (b) Immunohistochemistry was done on mouse colon tissue for studying protein expression and distribution. Real time PCR was performed to monitor relative expression levels of several transcripts under inflammatory conditions. (c) CFTR dependent chloride secretion was monitored using iodide efflux, chloride efflux and short circuit measurements in colonic epithelial cell line HT29CL19A. (d) Dextran sodium sulfate (DSS) induced colitis mouse model was used in Nherf2+/+ and Nherf2-/C57BL/6 male mice 7-8 weeks of age. Results: (a) Upregulation of iNOS increased CFTR channel function by at least two folds in In Vivo and In Vitro models of inflammation. The phenomenon involves cGMP dependent signaling mechanism. (b) CFTR interacts with iNOS mediated by NHERF2 in a PDZ domain-dependent manner. (c) Diarrhea is attenuated in Nherf2-/compared to Nherf2+/+ mice in DSS-induced colitis model which simulates IBD. Conclusion: (a) A macromolecular complex of CFTR, NHERF2 and iNOS is integrative to CFTR dependent diarrhea in IBD. This compartmentalization of proteins and consequently of the signaling effectors enables a rapid and fine-tuned regulation of CFTR function. (b) Such signaling modules that are part of pathological profile can be approached as targets for improvement of IBD conditions.