title: Alcohol increases the permeability of airway epithelial tight junctions in Beas-2B and NHBE cells.
abstract: BACKGROUND
  Tight junctions form a continuous belt-like structure between cells and act to regulate paracellular signaling. Protein kinase C (PKC) has been shown to regulate tight junction assembly and disassembly and is activated by alcohol. Previous research has shown that alcohol increases the permeability of tight junctions in lung alveolar cells. However, little is known about alcohol's effect on tight junctions in epithelium of the conducting airways. We hypothesized that long-term alcohol exposure reduces zonula occluden-1 (ZO-1) and claudin-1 localization at the cell membrane and increases permeability through a PKC-dependent mechanism.
  
  
  METHODS
  To test this hypothesis, we exposed normal human bronchial epithelial (NHBE) cells, cells from a human bronchial epithelial transformed cell line (Beas-2B), and Beas-2B expressing a PKCα dominant negative (DN) to alcohol (20, 50, and 100 mM) for up to 48 hours. Immunofluorescence was used to assess changes in ZO-1, claudin-1, claudin-5, and claudin-7 localization. Electric cell-substrate impedance sensing was used to measure the permeability of tight junctions between monolayers of NHBE, Beas-2B, and DN cells.
  
  
  RESULTS
  Alcohol increased tight junction permeability in a concentration-dependent manner and decreased ZO-1, claudin-1, claudin-5, and claudin-7 localization at the cell membrane. To determine a possible signaling mechanism, we measured the activity of PKC isoforms (alpha, delta, epsilon, and zeta). PKCα activity significantly increased in Beas-2B cells from 1 to 6 hours of 100 mM alcohol exposure, while PKCζ activity significantly decreased at 1 hour and increased at 3 hours. Inhibiting PKCα with Gö-6976 prevented the alcohol-induced protein changes in both ZO-1 and claudin-1 at the cell membrane. PKCα DN Beas-2B cells were resistant to alcohol-induced protein alterations.
  
  
  CONCLUSIONS
  These results suggest that alcohol disrupts ZO-1, claudin-1, claudin-5, and claudin-7 through the activation of PKCα, leading to an alcohol-induced "leakiness" in bronchial epithelial cells. Such alcohol-induced airway-leak state likely contributes to the impaired airway host defenses associated with acute and chronic alcohol ingestion.
authors.affiliation: Department of Internal Medicine, Pulmonary, Critical Care, Sleep & Allergy Division, University of Nebraska Medical Center, Omaha, Nebraska 68198-5910, USA.
authors.forename: Samantha M
authors.initials: SM
authors.lastname: Simet
authors.forename: Todd A
authors.initials: TA
authors.lastname: Wyatt
authors.forename: Jane
authors.initials: J
authors.lastname: DeVasure
authors.forename: Daniel
authors.initials: D
authors.lastname: Yanov
authors.forename: Diane
authors.initials: D
authors.lastname: Allen-Gipson
authors.forename: Joseph H
authors.initials: JH
authors.lastname: Sisson
mesh_terms: D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D002461:Cell Line, Transformed; D062445:Claudin-1; D062507:Claudin-5; D057167:Claudins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D000431:Ethanol / Q000008:administration & dosage / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D010750:Phosphoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000378:metabolism*; D019108:Tight Junctions / Q000187:drug effects* / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein
chemical_list: C568108:CLDN1 protein, human; C107466:CLDN5 protein, human; C473255:CLDN7 protein, human; D002227:Carbazoles; D062445:Claudin-1; D062507:Claudin-5; D057167:Claudins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C081021:Go 6976; D000431:Ethanol; D011493:Protein Kinase C