H. pylori stimulates ADAM 17 in epithelial cells independent of a functional cag PAI

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To extend results on H. pylori induced upregulation of ADAM17 transcripts in gastric epithelial cells [22], ADAM17 protein was quantified using “In Cell Western” analysis in AGS and MKN28 gastric epithelial cells and A431 cells following stimulation with the cag PAI+ H. pylori strain G27 and G27ΔcagM isogenic mutant. The 700nm Relative Value (Rel₇₀₀) of unstimulated controls for each cell line at both 1 and 3h time points (defined as 100% standard) was used for comparisons of changes in ADAM17 induced by H. pylori. In all three cell lines both G27 and the G27ΔcagM isogenic strain significantly increased ADAM17 in a time dependant manner (Figure 1A). Previous studies have demonstrated that G27ΔcagM fails to activate NF-κB [38] and IL-8 secretion in gastric epithelial cells [11]. The greatest increase in ADAM17 following H. pylori stimulation was observed in the AGS gastric epithelial cell line.

As ADAM17 is produced as a pro-protein which undergoes maturation and cell trafficking to the cell membrane where ADAM17 acts as a sheddase [39], “On Cell Western” assays [40] were used to assess the effects of H. pylori infection on surface ADAM17 in AGS cells. ADAM17 immunolabelling of the extracellular domain of the protein was carried out in unpermeabilised AGS cells and following permeabilisation, total cellular ERK1 was measured to control for cell density variations. Parallel assays of total cellular ADAM17 were undertaken by ICW on the same assay plates under identical experimental conditions. In the OCW both the wild type strain G27 and the ∆cagM mutant significantly increased surface cell membrane ADAM17 (Figure 1B). At 1h post-stimulation there was a 90% increase in surface ADAM17 relative to unstimulated control cells following G27 stimulation and 100% increase following G27ΔcagM (Figure 1B). Whilst there was a temporal increase in total cellular ADAM17 after bacterial stimulation as determined by the ICW assay over the course of the 6h experiment, the major increase in surface ADAM17 in the OCW assay was detected 1h post-stimulation with G27 and the isogenic mutant G27 ΔcagM (Figure 1B).