Occludin is essential for tumor necrosis factor (TNF)‐induced intestinal epithelial tight junction (TJ) disruption

TNF decreases intestinal epithelial TJ barrier function by a myosin light chain kinase- (MLCK-) dependent process. Although endocytosis of occludin following TNF-induced MLCK activation is required for barrier loss, specific contributions of occludin are poorly defined. Our aim was to define the role of occludin domains in TNF-induced TJ disruption. Stable occludin knockdown (KD) decreased transepithelial resistance (TER) of Caco-2 monolayers to 59 ± 7 % of wild type (WT). Analysis of cation and FITC-dextran flux showed that this was primarily due to increased permeability to large solutes; 3kDa FITC-dextran flux was 474 ± 52 % of control. Expression and trafficking of other TJ proteins were unaffected by occludin KD. TNF decreased TER of IFNγ-primed WT monolayers by 14 ± 1 % after 4 hrs, but TER of occludin KD monolayers was unchanged, despite increased MLC phosphorylation. Stable reexpression of WT occludin, but not a mutant lacking the C-terminal coiled-coil domain, resensitized occludin KD monolayers to TNF-induced barrier loss. This was not due to defective trafficking, as the occludin deletion mutant was efficiently delivered to the TJ. Thus, occludin contributes to TNF-induced TJ barrier loss by a mechanism that involves the C-terminal coiled-coil domain. This suggests that protein interactions mediated by the coiled-coil domain are required for TNF-induced barrier loss. Supported by NIH and CCFA.