Abstract
Occludin, claudin-1, claudin-2 and claudin-3 are membrane spanning proteins localized to the tight junction. These proteins are restricted to a discreet location within the membrane by a complex array of intracellular scaffolding proteins tethered to the cytoskeleton. Inflammatory disorders tend to disrupt tight junction protein complexes leading to impaired epithelial barrier function. The objective of this study was to examine the MDCK cell barrier function in response to the inflammatory cytokines tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ). Lactate dehydrogenase activity was used as a measure of cytotoxicity, transepithelial resistance (TER) and mannitol flux measurements were used to analyze barrier integrity, immunofluorescence and western blot analyses were used to analyze protein expression and localization. Exposure of MDCK cells to TNFα/IFNγ resulted in a marked and sustained elevation of TER as well as elevated paracellular permeability in a dose-dependent manner. Corresponding to increased TER and flux, we observed significant alterations in occludin, claudin-1 and claudin-2 protein expression, junctional localization and substantial cytoskeletal reorganization. Pharmacological inhibition of ERK1/2 and p38 signaling impaired the deleterious effects of the inflammatory cytokines on barrier function. These data strongly suggest that downstream effectors of MAP kinase signaling pathways mediate the TNFα/IFNγ-induced junctional reorganization that disrupts MDCK cell barrier function. Research funded by NIH Grant DK065652 and K.A. Dudowicz is a Merck/AAAS Scholar.
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