P-294 Inhibition of Protein Translation Initiation and Disruption of the Intestinal Epithelial Barrier in IBD

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Abstract

Background:

Integrity of the intestinal epithelial barrier is regulated by 2 types of adhesive structures: intercellular junctions and focal adhesions (FA) that control epithelial cell attachment to each other and to the extracellular matrix (ECM), respectively. Disassembly of adherens junctions (AJ) and tight junctions (TJ) plays key roles in the breakdown of the intestinal epithelial barrier during mucosal inflammation. Abnormal architecture and functions of FA significantly contribute to attenuated restitution of the inflamed intestinal mucosa. Proinflammatory cytokines such as interferon (IFN)-g and tumor necrosis factor (TNF)-α, are known to trigger AJ/TJ disassembly and attenuate wound healing in model intestinal epithelial cell monolayers. Mechanisms of cytokine-dependent downregulation of adhesion proteins remain poorly understood. Protein translation is an attractive mechanism, since it is known to be down-regulated by inflammatory stimuli in colonic and pancreatic epithelia. Initiation is a rate-limited step in protein translation and eIF4G1 and eIF4G2 are essential regulators of protein translation initiation with non-redundant functions. A little is known regarding the roles of eIF4G1 and eIF4G2 in the regulation of epithelial junctions and ECM adhesions.

Methods:

This study was performed using a well-differentiated clone of SC-CO15 and HT29 human colonic epithelial cell line (HT29cF8). Transient down-regulation of eIF4G1 and eIF4G2 was achieved using siRNA-mediated knock-down. Barrier function of epithelial cell monolayers was determined via transepithelial electrical resistance (TEER) measurements. Organization of AJ, TJ and FA in fixed cells was examined by immunofluorescence labeling and confocal microscopy.

Results:

Immunoblotting analysis of IFNg and IFNg/TNFα-treated intestinal epithelial cells demonstrated significant downregulation of AJ proteins, E-cadherin and p120 catenin, and the TJ protein, occludin. By contrast quantitative RT-PCR analysis did not reveal changes in mRNA levels of selected junctional proteins. Furthermore, expression of translation initiation factors eIF4G1 and eIF4G2 was downregulated by these proinflammatory cytokines in vitro, and eIF4G2 level was decreased in colonic mucosa of IBD patients. RNAi-dependent knockdown of either translation initiator resulted in the decreased TEER, indicating disruption of the paracellular barrier. By contrast, only eIF4G2 knockdown attenuated healing of intestinal epithelial wounds. Disruption of epithelial barrier in eIF4G1-depleted cells was accompanied by decreased expression of p120 catenin and E-cadherin and mislocalization of claudin-4 and β-catenin. Furthermore, immunoblotting analysis of major transmembrane, scaffolding, and signaling constituents of FA revealed that loss of eIF4G2 selectively decreased α-V integrin expression and paxillin phosphorylation without affecting the expression or phosphorylation of other proteins. Furthermore our latest experiments identified the leading edge as the hot-spot site of active translation during wound healing.

Conclusions:

These results reveal a novel role for protein translation initiation in regulating intestinal epithelial barrier and epithelial restitution and suggest that this important homeostatic mechanism could be impaired during mucosal inflammation and IBD.

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