Interferon-γ and Tumor Necrosis Factor-α Synergize to Induce Intestinal Epithelial Barrier Dysfunction by Up-Regulating Myosin Light Chain Kinase Expression

Abstract

Numerous intestinal diseases are characterized by immune cell activation and compromised epithelial barrier function. We have shown that cytokine treatment of epithelial monolayers increases myosin II regulatory light chain (MLC) phosphorylation and decreases barrier function and that these are both reversed by MLC kinase (MLCK) inhibition. The aim of this study was to determine the mechanisms by which interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha regulate MLC phosphorylation and disrupt epithelial barrier function. We developed a model in which both cytokines were required for barrier dysfunction. Barrier dysfunction was also induced by TNF-alpha addition to IFN-gamma-primed, but not control, Caco-2 monolayers. TNF-alpha treatment of IFN-gamma-primed monolayers caused increases in both MLCK expression and MLC phosphorylation, suggesting that MLCK is a TNF-alpha-inducible protein. These effects of TNF-alpha were not mediated by nuclear factor-kappaB. However, at doses below those needed for nuclear factor-kappaB inhibition, sulfasalazine was able to prevent TNF-alpha-induced barrier dysfunction, MLCK up-regulation, and MLC phosphorylation. Low-dose sulfasalazine also prevented morphologically evident tight junction disruption induced by TNF-alpha. These data show that IFN-gamma can prime intestinal epithelial monolayers to respond to TNF-alpha by disrupting tight junction morphology and barrier function via MLCK up-regulation and MLC phosphorylation. These TNF-alpha-induced events can be prevented by the clinically relevant drug sulfasalazine.

Prohibitin Inhibits Tumor Necrosis Factor alpha–induced Nuclear Factor-kappa B Nuclear Translocation via the Novel Mechanism of Decreasing Importin α3 Expression

“…Here we show that TNF-␣ decreases prohibitin mRNA and protein expression in a time-dependent manner. TNF-␣ decreases PHB mRNA and protein in a dose-dependent manner when using 1, 5, and 10 ng/ml TNF-␣, which are doses commonly used for TNF-␣ treatment of cultured intestinal epithelial cells (Ma et al, 2004;Wang et al, 2005), but not at 20 and 50 ng/ml TNF-␣. TNF-␣ is known to exude differing effects at lower (1-10 ng/ml) versus high doses (50 ng/ml), and perhaps at high doses it stimulates posttranscriptional modifications resulting in decreased PHB protein levels without a maximal decrease in mRNA.…”

“…PHB protein expression is significantly decreased after 8 h of TNF-␣ treatment and is sustained through 24 h ( Figure 1B). PHB expression was also measured in Caco2-BBE cells treated with increasing doses of TNF-␣ for 8 h. Although statistically significant only at 10 ng/ml, TNF-␣ decreases PHB mRNA ( Figure 1C) and protein ( Figure 1D) in a dose-dependent manner when using 1, 5, and 10 ng/ml TNF-␣, which are doses commonly used for TNF-␣ treatment of cultured intestinal epithelial cells (Ma et al, 2004;Wang et al, 2005). The effect of 20 or 50 ng/ml TNF-␣ on PHB mRNA expression is not as maximal as 10 ng/ml TNF-␣, whereas PHB protein is decreased to the same magnitude at 10, 20, or 50 ng/ml TNF-␣.…”

Epithelial myosin light chain kinase expression and activity are upregulated in inflammatory bowel disease

“…[13][14][15] In addition, we and others have recently shown that in vitro TNFinduced barrier dysfunction in model intestinal epithelia requires increased MLCK expression. 16,17 However, in vivo MLCK expression has not been examined in humans or experimental animals.…”

Inflammatory Bowel Disease Susceptibility Gene C1ORF106 Regulates Intestinal Epithelial Permeability

“…TNF-a inhibitors have been widely used in human IBD, with evidence of mucosal healing in patients that shows clinical benefit (16)(17)(18). We next asked whether C1ORF106 deficiency could exacerbate TNF-a-induced epithelial permeability.…”