Cellular and molecular mechanisms that mediate basal and tumour necrosis factor-alpha-induced regulation of myosin light chain kinase gene activity.
ABSTRACT The patients with Crohn's disease (CD) have a 'leaky gut' manifested by an increase in intestinal epithelial tight junction (TJ) permeability. Tumour necrosis factor-alpha (TNF-alpha) is a proto-typical pro-inflammatory cytokine that plays a central role in intestinal inflammation of CD. An important pro-inflammatory action of TNF-alpha is to cause a functional opening of intestinal TJ barrier. Previous studies have shown that TNF-alpha increase in TJ permeability was regulated by an increase in myosin light chain kinase (MLCK) gene activity and protein expression. The major aim of this study was to elucidate the cellular and molecular mechanisms that mediate basal and TNF-alpha-induced increase in MLCK gene activity. By progressive 5' deletion, minimal MLCK promoter was localized between -313 to +118 on MLCK promoter. A p53 binding site located within minimal promoter region was identified as an essential determinant for basal promoter activity. A 4 bp start site and a 5 bp downstream promoter element were required for MLCK gene activity. TNF-alpha-induced increase in MLCK promoter activity was mediated by NF-kappaB activation. There were eight kappaB binding sites on MLCK promoter. The NF-kappaB1 site at +48 to +57 mediated TNF-alpha-induced increase in MLCK promoter activity. The NF-kappaB2 site at -325 to -316 had a repressive role on promoter activity. The opposite effects on promoter activity were due to differences in the NF-kappaB dimer type binding to the kappaB sites. p50/p65 dimer preferentially binds to the NF-kappaB1 site and up-regulates promoter activity; while p50/p50 dimer preferentially binds to the NF-kappaB2 site and down-regulates promoter activity. In conclusion, we have identified the minimal MLCK promoter region, essential molecular determinants and molecular mechanisms that mediate basal and TNF-alpha-induced modulation of MLCK promoter activity in Caco-2 intestinal epithelial cells. These studies provide novel insight into the cellular and molecular mechanisms that regulate basal and TNF-alpha-induced modulation of MLCK gene activity.
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ABSTRACT: The ACCENT II study (A Crohn's Disease Clinical Trial Evaluating Infiximab in a New Long-term Treatment Regimen in Patients With Fistulizing Crohn's Disease) evaluated the efficacy and safety of infliximab maintenance treatment in patients with fistulizing Crohn's disease. This post hoc analysis was conducted to determine the efficacy and safety of infliximab therapy in women with rectovaginal fistulas. All patients received 5 mg/kg infliximab intravenously at weeks 0, 2, and 6. Patients who achieved response at weeks 10 and 14 then were randomized as responders if they had at least 50% of baseline fistulas closed, or as nonresponders, to receive placebo or infliximab 5 mg/kg every 8 weeks through week 54. Of 282 patients in the ACCENT II study, 25 of 138 (18.1%) women had a total of 27 draining rectovaginal fistulas at baseline. After infusions of infliximab at weeks 0, 2, and 6, 60.7% (17 of 28) and 44.8% (13 of 29) of rectovaginal fistulas were closed at weeks 10 and 14, respectively. Among responders, 72.2% (13 of 18) of rectovaginal fistulas were no longer draining at week 14. The duration of rectovaginal fistula closure was longer in the infliximab 5-mg/kg maintenance group (median, 46 wk) than in the placebo group (33 wk). Infliximab is effective in short-term closure of rectovaginal fistulas and maintenance treatment was more effective than placebo in prolonging rectovaginal fistula closure.Clinical Gastroenterology and Hepatology 11/2004; 2(10):912-20. · 6.65 Impact Factor
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ABSTRACT: TNF-alpha is a widely distributed proinflammatory cytokine, involved in many disease states. Although it has widely distributed effects, a precise mechanism of action has never been described, in particular at the epithelial level. Morpho-functional changes of the intestinal epithelial monolayer HT29 cl.19A exposed to TNF-alpha were therefore assessed, using electron microscopy (including freeze-fracture replica analysis), as well as measurement of mannitol, Na+ and horseradish peroxidase fluxes across intestinal HT29 cl.19A cell monolayers using Ussing chambers. TNF-alpha receptors were induced on HT29 cl.19A cells by a small non-toxic dose of IFN-gamma (5 U/ml). After 4 h of the combined presence of TNF-alpha (10 ng/ml) and IFN-gamma (5 U/ml), the tight junction structure was altered as shown by a significant decrease in the average strand number measured in the apico-basal direction (5.50 +/- 2.70 vs 3.73 +/- 1.39 in control and treated cells respectively, P < 0.0001) and by a significant decrease in junctional depth (0.27 +/- 0.14 and 0.17 +/- 0.10 microns in control and treated cells respectively, P < 0.0001). These results are in agreement with a decrease in number of 'kiss' sites between contiguous membranes of TNF-alpha treated cells observed in ultrathin sections.(ABSTRACT TRUNCATED AT 250 WORDS)Cytokine 07/1995; 7(5):441-8. · 2.52 Impact Factor
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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.American Journal Of Pathology 03/2005; 166(2):409-19. · 4.52 Impact Factor