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

Abstract

A TNF-alpha-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-alpha-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-alpha 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-alpha-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-alpha regulation of intestinal TJ barrier, we identified and cloned for the first time a functionally active MLCK promoter region. TNF-alpha 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-alpha-induced increase in MLCK transcription corresponded to a sequential increase in MLCK protein expression, MLCK activity, and Caco-2 TJ permeability. The TNF-alpha-induced increase in MLCK promoter activity was mediated by NF-kappaB activation, and the inhibition of NF-kappaB activation prevented the TNF-alpha-induced increase in promoter activity and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability. The TNF-alpha-induced activation of MLCK promoter was mediated by binding of the activated NF-kappaB p50/p65 dimer to the downstream kappaB binding site (-84 to -75) on the MLCK promoter region; deletion of the kappaB binding site prevented the TNF-alpha increase in promoter activity. Additionally, siRNA silencing of NF-kappaB p65 also prevented the TNF-alpha increase in MLCK promoter activity. In conclusion, our findings indicated that the TNF-alpha-induced increase in intestinal epithelial TJ permeability was mediated by NF-kappaB p50/p65 binding and activation of the MLCK promoter. NF-kappaB 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.

HIV-1 impairs human retinal pigment epithelial barrier function: possible association with the pathogenesis of HIV-associated retinopathy

“…In the presence of hr IL-6 and hr MCP-1, the expression of ZO-1 proteins was downregulated in D407 cells (Figures 5d and e), thereby impairing HRPE monolayer integrity (Figure 5c). TNF-a is often associated with viral infection and has been reported to impair the intestinal epithelial tight junction barrier; 45 however, in D407 cells we could not detect the release of TNF-a, possibly because different epithelial cells show distinct properties in response to viral infection. In addition, IL-6 and MCP-1 exerted no effect on the expressions of Occludin, Claudin-1, Claudin-2, Claudin-3, Claudin-4, or Claudin-5.…”

Tumor Necrosis Factor-induced Long Myosin Light Chain Kinase Transcription Is Regulated by Differentiation-dependent Signaling Events

“…Although Ma et al (11) also reported that TNF could induce increases in Caco-2 MLCK expression, they found that this up-regulation was blocked by NFB inhibition. Ma et al recently reported that the a single NFB binding site mediates a 50% increase in transcription in response to TNF (43). We have located this site ϳ150bp upstream of exon 1A, downstream of the 3Ј Sp1 site.…”

Evaluation the effect of thiamin deficiency on intestinal immunity of young grass carp (Ctenopharyngodon idella)

“…Additionally, thiamin deficiency elevated the mRNA level of MLCK may be partly due to the upregulation of pro-inflammatory cytokines mRNA levels. Studies had been shown that TNF-a [55] and IL-1b [56] can up-regulate the expression of MLCK in human Caco-2 cells. According to our results, correlation analysis revealed that the mRNA levels of MLCK were positively correlated with IFN-g2, TNF-a, IL-1b and IL-8 mRNA levels in young grass carp intestine, suggesting that dietary thiamin deficiency led to elevate the mRNA level of MLCK may be partly related to the up-regulated pro-inflammatory cytokines mRNA levels in fish.…”