title: Mechanisms of tumor necrosis factor-alpha-induced leaks in intestine epithelial barrier.
abstract: 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.
authors.affiliation: Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, Hunan 41008, China.
authors.forename: Fang
authors.initials: F
authors.lastname: He
authors.forename: Jing
authors.initials: J
authors.lastname: Peng
authors.forename: Xiao-lu
authors.initials: XL
authors.lastname: Deng
authors.forename: Li-fen
authors.initials: LF
authors.lastname: Yang
authors.forename: Aboubacar Damayé
authors.initials: AD
authors.lastname: Camara
authors.forename: Ahmed
authors.initials: A
authors.lastname: Omran
authors.forename: Guo-li
authors.initials: GL
authors.lastname: Wang
authors.forename: Li-wen
authors.initials: LW
authors.lastname: Wu
authors.forename: Ci-Liu
authors.initials: CL
authors.lastname: Zhang
authors.forename: Fei
authors.initials: F
authors.lastname: Yin
mesh_terms: D000199:Actins / Q000378:metabolism; D018938:Caco-2 Cells; D002463:Cell Membrane Permeability / Q000187:drug effects; D004848:Epithelium / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000187:drug effects* / Q000473:pathology*; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010957:Plasmids / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D000199:Actins; D020672:I-kappa B Proteins; D018994:Myosin Light Chains; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D034741:RNA, Small Interfering; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D009219:Myosin-Light-Chain Kinase