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

2006 ◽  
Vol 290 (3) ◽  
pp. G496-G504 ◽  
Author(s):  
Dongmei Ye ◽  
Iris Ma ◽  
Thomas Y. Ma
Keyword(s):  
Protein Expression ◽  
Tight Junction ◽  
Binding Site ◽  
Promoter Region ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Epithelial Tight Junction

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.

Mechanism of TNF-α modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression

2005 ◽  
Vol 288 (3) ◽  
pp. G422-G430 ◽  
Author(s):  
Thomas Y. Ma ◽  
Michel A. Boivin ◽  
Dongmei Ye ◽  
Ali Pedram ◽  
Hamid M. Said
Keyword(s):  
Protein Expression ◽  
Tight Junction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial ◽  
Mrna Transcription ◽  
Tnf Α ◽  
Epithelial Tight Junction

TNF-α plays a central role in the intestinal inflammation of various inflammatory disorders including Crohn's disease (CD). TNF-α-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed as one of the proinflammatory mechanisms contributing to the intestinal inflammation. The intracellular mechanisms involved in the TNF-α-induced increase in intestinal TJ permeability remain unclear. The purpose of this study was to investigate the possibility that the TNF-α-induced increase in intestinal epithelial TJ permeability was regulated by myosin light-chain kinase (MLCK) protein expression, using an in vitro intestinal epithelial model system consisting of the filter-grown Caco-2 intestinal epithelial monolayers. TNF-α (10 ng/ml) produced a time-dependent increase in Caco-2 MLCK expression. The TNF-α increase in MLCK protein expression paralleled the increase in Caco-2 TJ permeability, and the inhibition of the TNF-α-induced MLCK expression (by cycloheximide) prevented the increase in Caco-2 TJ permeability, suggesting that MLCK expression may be required for the increase in Caco-2 TJ permeability. The TNF-α increase in MLCK protein expression was preceded by an increase in MLCK mRNA expression but not an alteration in MLCK protein degradation. Actinomycin-D prevented the TNF-α increase in MLCK mRNA expression and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability, suggesting that the increase in MLCK mRNA transcription led to the increase in MLCK expression. The TNF-α increase in MLCK protein expression was also associated with an increase in Caco-2 MLCK activity. The cycloheximide inhibition of MLCK protein expression prevented the TNF-α increase in MLCK activity and Caco-2 TJ permeability. Moreover, inhibitors of MLCK, Mg2+-myosin ATPase, and metabolic energy prevented the TNF-α increase in Caco-2 TJ permeability, suggesting that the increase in MLCK activity was required for the TNF-α-induced opening of the Caco-2 TJ barrier. In conclusion, our results indicate for the first time that 1) the TNF-α increase in Caco-2 TJ permeability was mediated by an increase in MLCK protein expression, 2) the increase in MLCK protein expression was regulated by an increase in MLCK mRNA transcription, and 3) the increase in Caco-2 TJ permeability required MLCK protein expression-dependent increase in MLCK activity.

TNF-α-induced increase in intestinal epithelial tight junction permeability requires NF-κB activation

2004 ◽  
Vol 286 (3) ◽  
pp. G367-G376 ◽  
Author(s):  
Thomas Y. Ma ◽  
Gary K. Iwamoto ◽  
Neil T. Hoa ◽  
Vimesh Akotia ◽  
Ali Pedram ◽  
...  
Keyword(s):  
Protein Expression ◽  
Tight Junction ◽  
Etiologic Factor ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Abnormal Increase ◽  
Epithelial Tight Junction ◽  
Tight Junction Permeability ◽  
First Time

Crohn's disease (CD) patients have an abnormal increase in intestinal epithelial permeability. The defect in intestinal tight junction (TJ) barrier has been proposed as an important etiologic factor of CD. TNF-α increases intestinal TJ permeability. Because TNF-α levels are markedly increased in CD, TNF-α increase in intestinal TJ permeability could be a contributing factor of intestinal permeability defect in CD. Our purpose was to determine some of the intracellular mechanisms involved in TNF-α modulation of intestinal epithelial TJ permeability by using an in vitro intestinal epithelial system consisting of filter-grown Caco-2 monolayers. TNF-α produced a concentration- and time-dependent increase in Caco-2 TJ permeability. TNF-α-induced increase in Caco-2 TJ permeability correlated with Caco-2 NF-κB activation. Inhibition of TNF-α-induced NF-κB activation by selected NF-κB inhibitors, curcumin and triptolide, prevented the increase in Caco-2 TJ permeability, indicating that NF-κB activation was required for the TNF-α-induced increase in Caco-2 TJ permeability. This increase in Caco-2 TJ permeability was accompanied by down-regulation of zonula occludens (ZO)-1 proteins and alteration in junctional localization of ZO-1 proteins. TNF-α modulation of ZO-1 protein expression and junctional localization were also prevented by NF-κB inhibitors. TNF-α did not induce apoptosis in Caco-2 cells, suggesting that apoptosis was not the mechanism involved in TNF-α-induced increase in Caco-2 TJ permeability. These results demonstrate for the first time that TNF-α-induced increase in Caco-2 TJ permeability was mediated by NF-κB activation. The increase in permeability was associated with NF-κB-dependent downregulation of ZO-1 protein expression and alteration in junctional localization.

scholarly journals Matrix Metalloproteinase-9 (MMP-9) induced disruption of intestinal epithelial tight junction barrier is mediated by NF-κB activation

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0249544
Author(s):  
Rana Al-Sadi ◽  
Jessica Engers ◽  
Mohammad Haque ◽  
Steven King ◽  
Deemah Al-Omari ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Tight Junction ◽  
Intestinal Permeability ◽  
Barrier Function ◽  
Molecular Mechanisms ◽  
Intestinal Inflammation ◽  
Nuclear Translocation ◽  
Matrix Metalloproteinase 9 ◽  
Intestinal Epithelial ◽  
Metalloproteinase 9

Background Matrix Metalloproteinase-9 (MMP-9) has been shown to play a key role in mediating inflammation and tissue damage in inflammatory bowel disease (IBD). In patients with IBD, the intestinal tight junction (TJ) barrier is compromised as characterized by an increase in intestinal permeability. MMP-9 is elevated in intestinal tissue, serum and stool of patients with IBD. Previous studies from our laboratory showed that MMP-9 causes an increase in intestinal epithelial TJ permeability and that the MMP-9 induced increase in intestinal permeability is an important pathogenic factor contributing to the development of intestinal inflammation in IBD. However, the intracellular mechanisms that mediate the MMP-9 modulation of intestinal barrier function remain unclear. Aims The main aim of this study was to further elucidate the molecular mechanisms involved in MMP-9 induced increase in intestinal epithelial TJ permeability using Caco-2 monolayers as an in-vitro model system. Results MMP-9 induced increase in Caco-2 TJ permeability was associated with activation and cytoplasmic-to-nuclear translocation of NF-κB p65. Knocking-down NF-κB p65 by siRNA transfection prevented the MMP-9 induced expression of the NF-κB target gene IL-8, myosin light chain kinase (MLCK) protein expression, and subsequently prevented the increase in Caco-2 TJ permeability. In addition, the effect of MMP-9 on Caco-2 intestinal epithelial TJ barrier function was not mediated by apoptosis or necrosis. Conclusion Our data show that the MMP-9 induced disruption of Caco-2 intestinal epithelial TJ barrier function is regulated by NF-κB pathway activation of MLCK.

scholarly journals Mechanism of glucocorticoid regulation of the intestinal tight junction barrier

2007 ◽  
Vol 292 (2) ◽  
pp. G590-G598 ◽  
Author(s):  
Michel A. Boivin ◽  
Dongmei Ye ◽  
John C. Kennedy ◽  
Rana Al-Sadi ◽  
Chris Shepela ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Protein Expression ◽  
Tight Junction ◽  
Barrier Function ◽  
In Vitro Model ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Vitro Model

A defective intestinal epithelial tight junction (TJ) barrier has been proposed as an important pathogenic factor contributing to the intestinal inflammation of Crohn's disease. Glucocorticoids are first-line therapeutic agents for the treatment of moderate to severe Crohn's disease. Glucocorticoid treatment has been shown to induce retightening of the intestinal TJ barrier defect in Crohn's disease patients. However, the mechanisms that mediate the glucocorticoid therapeutic action on intestinal TJ barrier function remain unknown. The aim of this study was to elucidate the mechanism of glucocorticoid modulation of the intestinal epithelial TJ barrier using an in vitro model system. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro model to examine the effects of glucocorticoids on basal intestinal epithelial TJ barrier function and on TNF-α-induced disruption of the TJ barrier. Glucocorticoids (prednisolone and dexamethasone) did not have a significant effect on baseline Caco-2 TJ barrier function but prevented the TNF-α-induced increase in Caco-2 TJ permeability. The glucocorticoid protective effect against the TNF-α-induced increase in Caco-2 TJ permeability required activation of the glucocorticoid receptor (GR) complex. The activation of the GR complex resulted in GR complex binding to the glucocorticoid response element (GRE) site on DNA and activation of a GR-responsive promoter. Glucocorticoids inhibited the TNF-α-induced increase in myosin light chain kinase (MLCK) protein expression, a key process mediating the TNF-α increase in intestinal TJ permeability. The glucocorticoid inhibition of the TNF-α-induced increase in MLCK protein expression was due to the binding of the GR complex to a GRE binding site on the MLCK promoter region suppressing the TNF-α-induced activation. Glucocorticoids inhibit the TNF-α-induced increase in Caco-2 TJ permeability. The prednisolone protective action was mediated by binding of activated GR complex to the GRE site on the MLCK promoter, suppressing the TNF-α-induced increase in MLCK gene activity, protein expression, and subsequent opening of the intestinal TJ barrier.

Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability

2006 ◽  
Vol 290 (2) ◽  
pp. G204-G212 ◽  
Author(s):  
Karol Dokladny ◽  
Pope L. Moseley ◽  
Thomas Y. Ma
Keyword(s):  
Heat Stress ◽  
Protein Expression ◽  
Tight Junction ◽  
Heat Exposure ◽  
Protective Role ◽  
Intestinal Epithelial ◽  
Epithelial Tight Junction ◽  
Shock Proteins

The effects of physiologically relevant increase in temperature (37–41°C) on intestinal epithelial tight junction (TJ) barrier have not been previously studied. Additionally, the role of heat-shock proteins (HSPs) in the regulation of intestinal TJ barrier during heat stress remains unknown. Because heat-induced disturbance of intestinal TJ barrier could lead to endotoxemia and bacterial translocation during physiological thermal stress, the purpose of this study was to investigate the effects of modest, physiologically relevant increases in temperature (37–41°C) on intestinal epithelial TJ barrier and to examine the protective role of HSPs on intestinal TJ barrier. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro intestinal epithelial model system to assess the effects of heat exposure on intestinal TJ barrier. Exposure of filter-grown Caco-2 monolayers to modest increases in temperatures (37–41°C) resulted in a significant time- and temperature-dependent increases in Caco-2 TJ permeability. Exposure to modest heat (39 or 41°C) resulted in rapid and sustained increases in HSP expression; and inhibition of HSP expression produced a marked increase in heat-induced increase in Caco-2 TJ permeability ( P < 0.001). Heat exposure (41°C) resulted in a compensatory increase in Caco-2 occludin protein expression and an increase in junctional localization. Inhibition of HSP expression prevented the compensatory upregulation of occludin protein expression and produced a marked disruption in junctional localization of occludin protein during heat stress. In conclusion, our findings demonstrate for the first time that a modest, physiologically relevant increase in temperature causes an increase in intestinal epithelial TJ permeability. Our data also show that HSPs play an important protective role in preventing the heat-induced disruption of intestinal TJ barrier and suggest that HSP mediated upregulation of occludin expression may be an important mechanism involved in the maintenance of intestinal epithelial TJ barrier function during heat stress.

Sign in / Sign up

Export Citation Format

Share Document