Helicobacter pylori Dysregulation of Gastric Epithelial Tight Junctions by Urease-Mediated Myosin II Activation

doi:10.1053/j.gastro.2008.10.011

Gastroenterology

Volume 136, Issue 1, January 2009, Pages 236-246

https://doi.org/10.1053/j.gastro.2008.10.011 Get rights and content

Background & Aims

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.

Section snippets

Cell culture and reagents

MKN28 gastric epithelial cells were cultured at 37°C under 5% CO₂/95% O₂ in RPMI medium 1640 (GIBCO/BRL, Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Sigma, St Louis, MO) and gentamycin (20 μg/mL). To form monolayers, cells were grown on polyester Transwell (Sigma) permeable tissue culture inserts (0.4 μm pore size). The Rho-associated kinase inhibitor Y-27632 (Calbiochem, San Diego, CA) was used at a final concentration of 10 μM. Recombinant H pylori urease was obtained from Orovax

H pylori Alters Barrier Function in Gastric Epithelial Cells

MKN28 cells are human gastric epithelial cells that form confluent monolayers. To determine whether this was an appropriate model to investigate barrier function, we first established that MKN28 cells form functional tight junctions. MKN28 cells were grown to confluence and transepithelial electrical resistance (TER) was quantified. We found that as MKN28 cells reached confluence, there was a progressive increase in TER, indicating that these cells form functional tight junctions (Figure 1A).

Discussion

Previous investigations into mechanisms by which H pylori disrupts the tight junction complex have been limited by the lack of testable gastric epithelial cell models that form functional barriers. The present study has utilized a biologically relevant in vitro model of H pylori–gastric epithelial cell interactions to demonstrate that infection induces a progressive loss of TER which is attenuated by disruption of ureB and which is followed by disruption of occludin at the level of the tight

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: The authors disclose the following: Supported in part by National Institutes of Health grants DK 58587, DK 73902 and CA 77955 (R.M.P.), DK 48370 (L.A.P.), DK 061931 (J.R.T.), a Research Fellowship Award from the Crohn's and Colitis Foundation of America sponsored by Ms Laura McAteer Hoffman (L.S.), and The Vanderbilt Digestive Diseases Research Center (DK058404).

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Basic—Alimentary TractHelicobacter pylori Dysregulation of Gastric Epithelial Tight Junctions by Urease-Mediated Myosin II Activation

Background & Aims

Methods

Results

Conclusions

Section snippets

Cell culture and reagents

H pylori Alters Barrier Function in Gastric Epithelial Cells

Discussion

Gastroenterology

Curr Opin Microbiol

Am J Pathol

Pathol Res Pract

Gastroenterology

Lab Invest

J Biol Chem

J Biol Chem

Gastroenterology

Gastroenterology

Gastroenterology

Gastroenterology

Helicobacter infection and gastric neoplasia

J Pathol

Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach

Cancer Res

Helicobacter pylori and atrophic gastritis: importance of the cagA status

J Natl Cancer Inst

Effect of gastric pH on urease-dependent colonization of gnotobiotic piglets by Helicobacter pylori

Infect Immun

Purification of recombinant Helicobacter pylori urease apoenzyme encoded by ureA and ureB

Infect Immun

Cutting edge: urease release by Helicobacter pylori stimulates macrophage inducible nitric oxide synthase

J Immunol

Long-standing gastric mucosal barrier dysfunction in Helicobacter pylori-induced gastritis in Mongolian gerbils

Helicobacter

Basic—Alimentary Tract
Helicobacter pylori Dysregulation of Gastric Epithelial Tight Junctions by Urease-Mediated Myosin II Activation