Inflammatory bowel disease (IBD) affects more than 1 million Americans with more than 30,000 new cases diagnosed each year. IBD increases patient morbidity and susceptibility to colorectal cancer, yet its etiology remains unknown. Current models identify two key determinants of IBD pathogenesis: hyperpermeability of the gut epithelial barrier to bacterial products and an abnormal immune response to these products. Two factors seem critical for hyperpermeability: oxidant-induced stress and proinflammatory cytokines (e.g., tumor necrosis factor-␣). The aim of this study was to investigate the role of oxidant stressmediated transactivation of the epidermal growth factor receptor (EGFR) in intestinal hyperpermeability. This study used the Caco-2 human colonic epithelial cell in vitro model of intestinal epithelium. Cells were grown on inserts for permeability and signaling studies and glass coverslips for microscopy studies.We show that oxidant-induced intestinal hyperpermeability can be blocked by specific inhibitors of the EGFR, tumor necrosis factor convertase (TACE) metalloprotease, transforming growth factor (TGF)-␣, and mitogen-activated protein kinases, especially extracellular signal-regulated kinase 1/2. We also show that oxidant initiates these signaling events, in part by causing translocation of TACE to cell-cell contact zones. In this study, our data identify a novel mechanism for oxidant-induced intestinal hyperpermeability relevant to IBD. We propose a new intestinal permeability model in which oxidant transactivates EGFR signaling by activation of TACE and cleavage of precursor TGF-␣. These data could have a significant effect on our view of IBD pathogenesis and provide new therapeutic targets for IBD treatment.Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory disorder that affects more than 1 million Americans, with more than 30,000 new cases diagnosed each year (Podolsky, 2002;Farhadi et al., 2003;Clayburgh et al., 2004) This disorder profoundly increases patient morbidity and decreases quality of life and increases patient susceptibility to colorectal cancer, although its etiology remains unknown. Current models of disease pathogenesis identify as the two principal determinants of IBD pathogenesis: increased permeability of the epithelial barrier of the intestinal mucosa, which results in penetration of luminal bacterial products into the mucosa; and an abnormal immune response to these products (Farhadi et al., 2003;Clayburgh et al., 2004 ABBREVIATIONS: IBD, inflammatory bowel disease; TNF, tumor necrosis factor; TJ, tight junction; IEC, intestinal epithelial cell; EGFR, epidermal growth factor receptor; PKC, protein kinase C; EGF, epidermal growth factor; TGF, transforming growth factor; TACE, tumor necrosis factor-␣-converting enzyme; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; TAPI-2, , 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; GW2974, N4-(1-benzyl-1H-indazol-5-yl)-N 6 ,N 6 -dimethyl-pyrido[3,4-D]pyrimidine-4,6-diamine; ...

Using monolayers of intestinal Caco-2 cells, we discovered that the isoform of protein kinase C (PKC), a member of the "novel" subfamily of PKC isoforms, is required for monolayer barrier function. However, the mechanisms underlying this novel effect remain largely unknown. Here, we sought to determine whether the mechanism by which PKC-disrupts monolayer permeability and dynamics in intestinal epithelium involves PKC--induced alterations in claudin isotypes. We used cell clones that we recently developed, clones that were transfected with varying levels of plasmid to either stably suppress endogenous PKC-activity (antisense, dominant-negative constructs) or to ectopically express PKC-activity (sense constructs). We then determined barrier function, claudin isotype integrity, PKC-subcellular activity, claudin isotype subcellular pools, and claudin phosphorylation. Antisense transfection to underexpress the PKC-led to monolayer instability as shown by reduced 1) endogenous PKC-activity, 2) claudin isotypes in the membrane and cytoskeletal pools (2claud-1, 2claud-4 assembly), 3) claudin isotype phosphorylation (2 phospho-serine, 2 phospho-threonine), 4) architectural stability of the claudin-1 and claudin-4 rings, and 5) monolayer barrier function. In these antisense clones, PKC-activity was also substantially reduced in the membrane and cytoskeletal cell fractions. In wild-type (WT) cells, PKC-(82 kDa) was both constitutively active and coassociated with claudin-1 (22 kDa) and claudin-4 (25 kDa), forming endogenous PKC-/claudin complexes. In a second series of studies, dominant-negative inhibition of the endogenous PKC-caused similar destabilizing effects on monolayer barrier dynamics, including claudin-1 and -4 hypophosphorylation, disassembly, and architectural instability as well as monolayer disruption. In a third series of studies, sense overexpression of the PKC-caused not only a mostly cytosolic distribution of this isoform (i.e., Ͻ12% in the membrane ϩ cytoskeletal fractions, indicating PKC-inactivity) but also led to disruption of claudin assembly and barrier function of the monolayer. The conclusions of this study are that PKC-activity is required for normal claudin assembly and the integrity of the intestinal epithelial barrier. These effects of PKC-are mediated at the molecular level by changes in phosphorylation, membrane assembly, and/or organization of the subunit components of two barrier function proteins: claudin-1 and claudin-4 isotypes. The ability of PKC-to alter the dynamics of permeability protein claudins is a new function not previously ascribed to the novel subfamily of PKC isoforms.The epithelium of the intestinal mucosa is the largest interface between the body and the external environment. An important characteristic of this interface is its ability to maintain a highly selective permeability barrier that protects the internal milieu from hostile factors in the lumenal environment. Barrier permeability, which in general is maintained by epithelial tight-junctional proteins, pe...