Mechanisms of tumor necrosis factor-alpha-induced leaks in intestine epithelial barrier.

Objective: The aim of this study was to investigate the signaling mechanisms surrounding changes in tight junction (TJ) and the permeability of human intestinal epithelial cell induced by tumor necrosis factor-alpha (TNF-α).

Methods: To confirm that TNF-α induces epithelial barrier hyperpermeability by disrupting tight junction, Caco-2 cells were exposed to TNF-α, and changes in epithelial permeability (via TER assay), F-actin dynamics (via Rhodamine-phalloidin staining) and tight junction protein expression (via western blot) were monitored. Moreover, to ensure that NF-κB participated in the regulatory mechanisms, Caco-2 cells were transfected with DNMu-IκBα or control plasmids, the above experiments were repeated and the activation effect of TNF-α on NF-κB was detected by luciferase reporter assays. Lastly, we took dominant negative plasmid and knockdown approaches to investigate the potential importance of the NF-κB/myosin light chain kinase (MLCK)/myosin light chain phosphorylation (pMLC) pathways in TNF-a-mediated damage.

Results: TNF-α could cause NF-κB activation, F-actin rearrangement, tight junction disruption and barrier dysfunction. These effects were alleviated by inhibiting NF-κB. TNF-α induced increase of MLCK transcription and MLC phosphorylation act later than NF-κB activation, which could be suppressed both by inactivating and deleting NF-κB.

Conclusions: TNF-α induces intestinal epithelial cell hyperpermeability by disrupting TJs, in part through MLCK upregulation, in which NF-κB is the positive upstream regulator for MLCK.