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HELICOBACTER PYLORI INDUCES CALPAIN-DEPENDENT TRUNCATION AND REDISTRIBUTION OF E-CADHERIN IN EPITHELIAL MONOLAYERS
PM O’Connor1, TK Lapointe1, AG Buret1
1Department of Biological Sciences and Inflammation Research Network, University of Calgary, Calgary, Alberta
H.pylori colonizes 50% of the human population and is a risk factor for gastritis, gastroduodenal ulcers, and gastric adenocarcinoma. The ability of H.pylori to disrupt intercellular adhesion may influence disease severity. Alterations to the adherens junction protein E-cadherin have a well-established role in carcinogenesis. Members of the calpain family of cysteine proteases are upregulated in several types of adenocarcinoma and are able to cleave E-cadherin to a functionally-impaired 100kDa form.
AIM: The aim of this study was to determine the effects of H.pylori on E-cadherin and assess the role of calpain in these effects.
METHODS: Confluent non-tumorigenic epithelial cell (SCBN) monolayers were pre-treated or not with the selective calpain inhibitor PD150606 (50 µM) for 15 minutes. Cells were then sham treated or challenged with H.pylori strain SS1 or 60190 for 24h at an MOI of 100:1 and processed for immocytochemistry, western blotting, or enzyme activity assays.
RESULTS: H.pylori challenged monolayers displayed increased calpain activity and elevated levels of the cleaved form of the calpain substrate alpha-spectrin, as determined by activity assay and immunoblotting, respectively. Immunoblotting for the µ and m calpain isoforms revealed that µ, but not m, calpain is activated in response to H.pylori. Immunocytochemistry and western blotting of subcellular fractions revealed loss of membrane -associated E-cadherin and increased cytosolic E-cadherin. Western blotting of whole cell lysates demonstrated that H.pylori challenge resulted in cleavage of the 120kDa full-length E-cadherin to a 100kDa form. Pre-treatment with a calpain inhibitor prevented these junctional defects.
CONCLUSION: H.pylori induces isoform-specific calpain activation which results in cleavage and re-distribution of E-cadherin in non-tumorigenic epithelial cells. Future studies will evaluate the bacterial factors involved in these effects and the role of this disruption in H.pylori-induced cellular migration.