Molecular mechanism of tumor necrosis factor-α modulation of intestinal epithelial tight junction barrier

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Abstract
A TNF-α-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed to be an important proinflammatory mechanism contributing to intestinal inflammation in Crohn's disease and other inflammatory conditions. Previous studies from our laboratory suggested that the TNF-α-induced increase in intestinal TJ permeability was mediated by an increase in myosin light chain kinase (MLCK) protein expression. However, the molecular mechanisms that mediate the TNF-α increase in intestinal TJ permeability and MLCK protein expression remain unknown. The purpose of this study was to delineate the intracellular and molecular mechanisms that mediate the TNF-α-induced increase in intestinal TJ permeability; using an in vitro intestinal epithelial model system consisting of filter-grown Caco-2 intestinal epithelial monolayers. To examine the molecular mechanisms involved in the TNF-α regulation of intestinal TJ barrier, we identified and cloned for the first time a functionally active MLCK promoter region. TNF-α treatment of filter-grown Caco-2 monolayers transfected with plasmid vector containing the MLCK promoter region produced an increase in MLCK promoter activity and MLCK transcription. The TNF-α-induced increase in MLCK transcription corresponded to a sequential increase in MLCK protein expression, MLCK activity, and Caco-2 TJ permeability. The TNF-α-induced increase in MLCK promoter activity was mediated by NF-κB activation, and the inhibition of NF-κB activation prevented the TNF-α-induced increase in promoter activity and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability. The TNF-α-induced activation of MLCK promoter was mediated by binding of the activated NF-κB p50/p65 dimer to the downstream κB binding site (−84 to −75) on the MLCK promoter region; deletion of the κB binding site prevented the TNF-α increase in promoter activity. Additionally, siRNA silencing of NF-κB p65 also prevented the TNF-α increase in MLCK promoter activity. In conclusion, our findings indicated that the TNF-α-induced increase in intestinal epithelial TJ permeability was mediated by NF-κB p50/p65 binding and activation of the MLCK promoter. NF-κB p50/p65 activation of the MLCK promoter then leads to a stepwise increase in MLCK transcription, expression and activity, and MLCK-mediated opening of the intestinal TJ barrier.

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