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AMP-activated Protein Kinase Mediates the Interferon

AMP-activated Protein Kinase Mediates the Interferon-?-induced Decrease in Intestinal Epithelial Barrier Function* Received for publication,July20,2009,and in revised form,August3,2009Published,JBC Papers in Press,August4,2009,DOI10.1074/jbc.M109.046292 Michael Scharl,Gisela Paul,Kim E.Barrett,and Declan F.McCole1

From the Department of Medicine,University of California,San Diego,School of Medicine,La Jolla,California92093

Impaired epithelial barrier function plays a crucial role in the pathogenesis of inflammatory bowel disease.Elevated levels of the pro-inflammatory cytokine,interferon-?(IFN?),are believed to be prominently involved in the pathogenesis of Crohn disease.Treatment of T84intestinal epithelial cells with IFN?severely impairs their barrier properties measured as tran-sepithelial electrical resistance(TER)or permeability and reduces the expression of tight junction proteins such as occlu-din 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 IFN?-induced effects on the intestinal epithelial bar-rier.We found that IFN?activates AMPK by phosphorylation, independent of intracellular energy levels.Inhibition of AMPK prevents,at least in part,the IFN?-induced decrease in TER. Furthermore,AMPK knockdown prevented the increased epi-thelial permeability,the decreased TER,and the decrease in occludin and ZO-1caused by IFN?treatment of T84cells.How-ever,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 IFN?,in medi-ating 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. Inflammatory bowel disease(IBD)2consists of two major subgroups,ulcerative colitis and Crohn disease(CD).A com-plex cascade of genetic,immunological,and bacterial factors contributes to IBD pathogenesis(1).In the healthy intestine,the epithelial barrier separates the luminal bacterial microbiota and other aspects of the external environment from cells of the mucosal immune system.In CD in particular,an impaired epi-thelial barrier(2,3)leads to increased exposure of the immune system to commensal bacteria.Along with possible genetic defects in bacterial sensing,this might contribute to a dysregu-lated immune response leading to further epithelial damage and active episodes of IBD(4).Epithelial barrier dysfunction in CD is characterized by alterations in intercellular tight junc-tions(5),as well as by an excessive loss of water and salt into the lumen.An important immunological marker in CD is the exist-ence of excessively high levels of the pro-inflammatory cyto-kine,interferon gamma(IFN?)(6).

IFN?treatment of intestinal epithelial cell monolayers severely compromises their barrier integrity.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.This effect is due to disruption of the apical actin cytoskeleton in con-junction with decreased expression,as well as increased internal-ization,of important tight junction proteins such as occludin and zonula occludens-1(ZO-1)(8–11).Conversely,induction of epi-thelial apoptosis by IFN?is believed to contribute little to barrier dysfunction(12).IFN?also induces further alterations in epithelial function that include reduced expression of various ion transport-ers and associated decreases in epithelial ion transport(13,14). Despite the influence of IFN?on a number of epithelial functions, relatively little is known about intracellular signaling mechanisms mediating its effects following receptor activation.Recent studies demonstrated the involvement of phosphatidylinositol3?-kinase (PI3K)in mediating IFN?-induced effects on epithelial barrier function(11,15).However,this is unlikely to be the only regulatory pathway involved.Indeed,increased expression of receptors for tumor necrosis factor core family members,such as the tumor necrosis factor receptor and LIGHT(homologous to lympho-toxin,shows inducible expression and competes with herpes sim-plex virus glycoprotein D for herpes virus entry mediator(HVEM), a receptor expressed by T lymphocytes),can also occur in response to IFN?and lead to changes in intestinal barrier function(16–18). The effects of IFN?in intestinal epithelial cells resemble,at least in part,those of the cellular energy sensor,AMP-activated protein kinase(AMPK).Upon activation,AMPK restores intra-cellular ATP levels by stimulating energy-producing pathways, such as glucose uptake(19)and glycolysis,while inhibiting energy-consuming pathways,such as the synthesis of fatty acids or triglycerides(20,21).In the intestine,energy-consuming processes include epithelial ion transport,and,indeed,AMPK

*This work was supported,in whole or in part,by National Institutes of Health Grant DK080506.This work was also supported by a Career Development Award and a Senior Research Award from the Crohn’s and Colitis Founda-tion of America(to D.F.M.),scholarships from the German Research Foun-dation(Deutsche Forschungsgemeinschaft)(to M.S.and G.P.),and by the UCSD Digestive Diseases Research Development Center.

1To whom correspondence should be addressed:Division of Gastroenterol-ogy,University of California,San Diego,9500Gilman Drive,La Jolla,CA 92093-0063.Tel.:858-534-2794;Fax:858-534-3338;E-mail:dmccole@ ucsd.edu.

2The abbreviations used are:IBD,inflammatory bowel disease;CD,Crohn disease;PBS,phosphate-buffered saline;CC,Compound C;AMPK,AMP-activated protein kinase;Akt,protein kinase B;FITC,fluorescein isothiocya-nate;IFN?,interferon?;LY294002,2-(4-morpholinyl)-8-phenyl-1(4H)-ben-zopyran-4-one hydrochloride;PI3K,phosphatidylinositol3?-kinase;siRNA, small interfering RNA;TER,transepithelial electrical resistance;ZO-1, zonula occludens-1.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.284,NO.41,pp.27952–27963,October9,2009©2009by The American Society for Biochemistry and Molecular Biology,Inc.Printed in the U.S.A.

27952JOURNAL OF BIOLOGICAL

AMP-activated Protein Kinase Mediates the Interferon

CHEMISTRY VOLUME284?NUMBER41?OCTOBER9,2009

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