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HELICOBACTER PYLORI-INDUCED E-CADHERIN DISRUPTION IN NON-TUMORIGENIC EPITHELIAL MONOLAYERS IS MLCK-DEPENDENT

PM O'Connor, TK Lapointe, JP Fedwick, AG Buret
Department of Biological Sciences and Inflammation Research Network, University of Calgary, Calgary, Alberta

H.pylori (Hp) infection causes gastritis, gastroduodenal ulcers and gastric adenocarcinoma via mechanisms that remain obscure. The ability of Hp to alter intercellular adhesion may contribute to the development of serious disease. Disruption of adherens junction protein E-cadherin has been implicated in carcinogenesis and it was recently shown that Hp can directly alter tight junction integrity in gastric epithelia. The effects of Hp on E-cadherin, and whether such changes may involve a known regulator of epithelial barrier function, myosin-light chain kinase (MLCK), are unknown.
AIM: The aim of this study was to characterize the effects of Hp on E-cadherin in non-tumorigenic epithelial monolayers, and to examine the role of MLCK in these effects.
METHODS: This study applied a previously validated model using a non-tumorigenic epithelial cell line (SCBN) challenged with Hp strain SS1 or the human isolate 60190 (ATCC). Confluent Hp-infected or sham-treated monolayers grown in chamber slides were immunostained for E-cadherin. Western blot analysis was performed on cell lysates from sham treated or Hp-infected monolayers, pre-treated or not with a selective MLCK inhibitor (ML-9). Blots were probed for myosin light chain (MLC), phospho-myosin light chain 2 (MLC-2) and E-cadherin.
RESULTS: Hp increased levels of phosphorylated MLC while MLC-2 levels remained unchanged. Immunocytochemistry revealed the presence of cytosolic E-cadherin in Hp infected monolayers, but not in controls. Immunoblots demonstrated E-cadherin cleavage by the appearance of an E-cadherin fragment of approximately 50kDa after 12h infection. MLCK inhibition prevented this effect.
CONCLUSION: Hp-induced phosphorylation of epithelial MLC is associated with disruptions of E-cadherin in non-tumorigenic epithelial cells. These alterations of E-cadherin are regulated, at least in part, by MLCK. Future studies will examine whether MLCK-mediated disruption of epithelial barrier integrity is implicated in Hp-induced carcinogenesis.

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