title: Helicobacter pylori dysregulation of gastric epithelial tight junctions by urease-mediated myosin II activation.
abstract: OBJECTIVE
  Helicobacter pylori-induced gastritis predisposes to the development of gastric cancer. Increased epithelial tight junction permeability and alterations in apical-junctional complexes are also associated with an increased risk of carcinogenesis. Phosphorylation of myosin regulatory light chain (MLC) by MLC kinase (MLCK) regulates tight junction function. We determined whether MLCK was activated by H pylori and defined the mechanisms through which such activation dysregulates gastric epithelial barrier function.
  
  
  METHODS
  MKN28 gastric epithelial cells were cocultured with the H pylori cag(+) strain 60190 or cagA(-), cagE(-), ureB(-), or vacA(-) mutants. MLC phosphorylation and barrier integrity were determined by immunoblot analysis and transepithelial electrical resistance measurements, respectively. Localization of the tight junction protein occludin was determined by immunocytochemistry in MKN28 cells and INS-GAS mice.
  
  
  RESULTS
  H pylori induced a progressive loss of barrier function that was attenuated by inactivation of ureB, but not cagA, cagE, or vacA. Reductions in transepithelial electrical resistance were also dependent on functional urease activity. H pylori increased MLC phosphorylation in epithelial monolayers; this was significantly decreased by inhibition of MLCK or Rho kinase or by loss of UreB. H pylori infection of either cultured monolayers or hypergastrinemic INS-GAS mice induced occludin endocytosis, reflecting cytoskeletally mediated disruption of tight junctions.
  
  
  CONCLUSIONS
  H pylori increases MLC phosphorylation, occludin internalization and barrier dysfunction in gastric epithelial cells. This process requires functional urease activity and is independent of the cag pathogenicity island or VacA. These data provide new insights into the mechanisms by which H pylori disrupts gastric barrier function.
authors.affiliation: Division of Gastroenterology, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2279, USA.
authors.forename: Lydia E
authors.initials: LE
authors.lastname: Wroblewski
authors.forename: Le
authors.initials: L
authors.lastname: Shen
authors.forename: Seth
authors.initials: S
authors.lastname: Ogden
authors.forename: Judith
authors.initials: J
authors.lastname: Romero-Gallo
authors.forename: Lynne A
authors.initials: LA
authors.lastname: Lapierre
authors.forename: Dawn A
authors.initials: DA
authors.lastname: Israel
authors.forename: Jerrold R
authors.initials: JR
authors.lastname: Turner
authors.forename: Richard M
authors.initials: RM
authors.lastname: Peek
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D017097:Electric Impedance; D005753:Gastric Mucosa / Q000737:chemistry / Q000378:metabolism*; D016480:Helicobacter pylori / Q000201:enzymology / Q000472:pathogenicity*; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D018994:Myosin Light Chains / Q000378:metabolism; D024462:Myosin Type II / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D062793:Occludin; D010766:Phosphorylation; D019108:Tight Junctions / Q000502:physiology*; D014510:Urease / Q000502:physiology*; D054460:rho-Associated Kinases / Q000502:physiology
chemical_list: D008565:Membrane Proteins; D018994:Myosin Light Chains; D062793:Occludin; C568986:Ocln protein, mouse; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; D014510:Urease; D024462:Myosin Type II