title: Rebeccamycin Attenuates TNF-α-Induced Intestinal Epithelial Barrier Dysfunction by Inhibiting Myosin Light Chain Kinase Production.
abstract: OBJECTIVE
  Although proinflammatory cytokine-induced disruption of intestinal epithelial barrier integrity is associated with intestinal inflammatory disease, effective treatment for barrier dysfunction is lacking. Previously, we demonstrated that rebeccamycin alleviates epithelial barrier dysfunction induced by inflammatory cytokines in Caco-2 cell monolayers; however, the underlying mechanism remained unclear. Here, we investigated the mechanism by which rebeccamycin protects the epithelial barrier function of Caco-2 cells exposed to TNF-α.
  
  
  METHODS
  To confirm the epithelial barrier function of Caco-2 cell monolayers, transepithelial electrical resistance (TER) and paracellular permeability were measured. Production levels and localization of tight junction (TJ) proteins were analyzed by immunoblot and immunofluorescence, respectively. Phosphorylated myosin light chain (pMLC) and MLC kinase (MLCK) mRNA expression levels were determined by immunoblot and quantitative RT-PCR, respectively.
  
  
  RESULTS
  Rebeccamycin attenuated the TNF-α-induced reduction in TER and increase in paracellular permeability. Rebeccamycin increased claudin-5 expression, but not claudin-1, -2, -4, occludin or ZO-1 expression, and prevented the TNF-α-induced changes in ZO-1 and occludin localization. Rebeccamycin suppressed the TNF-α-induced increase in MLCK mRNA expression, thus suppressing MLC phosphorylation. The rebeccamycin-mediated reduction in MLCK production and protection of epithelial barrier function were alleviated by Chk1 inhibition.
  
  
  CONCLUSIONS
  Rebeccamycin attenuates TNF-α-induced disruption of intestinal epithelial barrier integrity by inducing claudin-5 expression and suppressing MLCK production via Chk1 activation.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Watari
authors.forename: Yuta
authors.initials: Y
authors.lastname: Sakamoto
authors.forename: Kota
authors.initials: K
authors.lastname: Hisaie
authors.forename: Kazuki
authors.initials: K
authors.lastname: Iwamoto
authors.forename: Miho
authors.initials: M
authors.lastname: Fueta
authors.forename: Kiyohito
authors.initials: K
authors.lastname: Yagi
authors.forename: Masuo
authors.initials: M
authors.lastname: Kondoh
mesh_terms: D018938:Caco-2 Cells; D002227:Carbazoles / Q000494:pharmacology*; D000071877:Checkpoint Kinase 1 / Q000378:metabolism; D062507:Claudin-5 / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology*; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000096:biosynthesis*; D010766:Phosphorylation / Q000187:drug effects; D019108:Tight Junctions / Q000201:enzymology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: C107466:CLDN5 protein, human; D002227:Carbazoles; D062507:Claudin-5; D018994:Myosin Light Chains; D014409:Tumor Necrosis Factor-alpha; C053339:rebeccamycin; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D009219:Myosin-Light-Chain Kinase
keywords: Checkpoint kinase 1; Epithelial barrier; Intestinal cells; Myosin light chain kinase; TNF-α; Tight junction