Abstract
Impaired epithelial barrier function plays a crucial role in the pathogenesis of inflammatory bowel disease. Elevated levels of the pro-inflammatory cytokine, interferon-gamma (IFNgamma), are believed to be prominently involved in the pathogenesis of Crohn disease. Treatment of T(84) intestinal epithelial cells with IFNgamma severely impairs their barrier properties measured as transepithelial electrical resistance (TER) or permeability and reduces the expression of tight junction proteins such as occludin and zonula occludens-1 (ZO-1). However, little is known about the signaling events that are involved. The cellular energy sensor, AMP-activated protein kinase (AMPK), is activated in response to cellular stress, as occurs during inflammation. The aim of this study was to investigate a possible role for AMPK in mediating IFNgamma-induced effects on the intestinal epithelial barrier. We found that IFNgamma activates AMPK by phosphorylation, independent of intracellular energy levels. Inhibition of AMPK prevents, at least in part, the IFNgamma-induced decrease in TER. Furthermore, AMPK knockdown prevented the increased epithelial permeability, the decreased TER, and the decrease in occludin and ZO-1 caused by IFNgamma treatment of T(84) cells. However, AMPK activity alone was not sufficient to cause alterations in epithelial barrier function. These data show a novel role for AMPK, in concert with other signals induced by IFNgamma, in mediating reduced epithelial barrier function in a cell model of chronic intestinal inflammation. These findings may implicate AMPK in the pathogenesis of chronic intestinal inflammatory conditions, such as inflammatory bowel disease.
Highlights
IFN␥ Activates AMPK in Human T84 Intestinal Epithelial Cells—To study whether the cellular energy sensor, AMPK, participates in IFN␥-induced effects on epithelial barrier function, we initially investigated whether IFN␥ activates AMPK
Representative Western blots showed that IFN␥ stimulated AMPK activation as early as 30 min after treatment; thereafter phosphorylation increased in a time-dependent manner reaching a peak at 6 h (Fig. 1A)
IFN␥-induced AMPK Activation Is Independent of Changes in Intracellular ATP—Because AMPK is usually activated by declining levels of intracellular ATP, we investigated if AMPK activation in response to IFN␥ was dependent on the intracellular ATP level
Summary
Most importantly from a functional perspective, IFN␥ causes a decrease in transepithelial electrical resistance (TER) and increases epithelial permeability [7, 8] These defects closely resemble observations in CD, where there is a disruption of intercellular tight junctional complexes. Recent studies demonstrated the involvement of phosphatidylinositol 3Ј-kinase (PI3K) in mediating IFN␥-induced effects on epithelial barrier function [11, 15]. This is unlikely to be the only regulatory pathway involved. IFN␥-induced Epithelial Barrier Dysfunction Involves AMPK has been shown to decrease intestinal ATP-consuming ion transport as well as the synthesis of various proteins [22, 23]. It has previously been demonstrated that ion transport processes are suppressed in intestinal biopsies from IBD patients (24 –26)
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