Abstract
BackgroundThe tight junction is a dynamic structure that is regulated by a number of cellular signaling processes. Occludin, claudin-1, claudin-2 and claudin-3 are integral membrane proteins found in the tight junction of MDCK cells. These proteins are restricted to this region of the membrane by a complex array of intracellular proteins which are tethered to the cytoskeleton. Alteration of these tight junction protein complexes during pathological events leads to impaired epithelial barrier function that perturbs water and electrolyte homeostasis. We examined MDCK cell barrier function in response to challenge by the proinflammatory cytokines tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ).ResultsExposure of MDCK cells to TNFα/IFNγ resulted in a marked sustained elevation of transepithelial electrical resistance (TER) as well as elevated paracellular permeability. We demonstrate that the combination of TNFα/IFNγ at doses used in this study do not significantly induce MDCK cell apoptosis. 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 blocked the deleterious effects of the proinflammatory cytokines on barrier function.ConclusionThese data strongly suggest that downstream effectors of MAP kinase signaling pathways mediate the TNFα/IFNγ-induced junctional reorganization that modulates MDCK cell barrier function.
Highlights
The tight junction is a dynamic structure that is regulated by a number of cellular signaling processes
In a variety of disease states inflammation is thought to negatively impact epithelial barrier function, we report that tumor necrosis factor-α (TNFα)/IFNγ co-administration to MDCK cell monolayers impaired epithelial barrier function as measured by elevated paracellular flux and produced marked elevation in transepithelial electrical resistance (TER)
In order to determine whether TNFα/IFNγ induced cytotoxic effects in the MDCK cell cultures, we determined the percentage of apoptotic cells in confluent MDCK cultures using the TUNEL assay (Table 1) and measured Lactate dehydrogenase (LDH) enzyme activity (Figure 1A) released from treated confluent cultures
Summary
The tight junction is a dynamic structure that is regulated by a number of cellular signaling processes. Claudin-1, claudin-2 and claudin-3 are integral membrane proteins found in the tight junction of MDCK cells. These proteins are restricted to this region of the membrane by a complex array of intracellular proteins which are tethered to the cytoskeleton. Alteration of these tight junction protein complexes during pathological events leads to impaired epithelial barrier function that perturbs water and electrolyte homeostasis. BMC Physiology 2006, 6:2 http://www.biomedcentral.com/1472-6793/6/2 molecular movement within the paracellular space These tight junction proteins form a dynamic seal between epithelial cells becoming the principle physical paracellular barrier. MDCK II cells express claudin-1, -2, -3 and -4 [7], the expression of claudin-2 in the MDCK II cells is in part responsible for its low electrical resistance profile [8]
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