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Helicobacter pylori colonizes the human stomach by infecting gastric epithelial cells. It is the primary cause of peptic ulcer disease and gastric cancer (GC). Cytotoxin-associated gene A (CagA) is a virulence factor produced by H. pylori. Strains positive for the CagA protein are associated with more severe gastric diseases. The 3' region of the cagA gene exhibits heterogeneity with respect to tyrosine phosphorylation motifs (EPIYA) and CagA multimerization motifs (CM). CagA proteins are categorized as either Western or Eastern based on EPIYA sequences. CM motifs are also identified as Western and Eastern based on CM sequences identified in Western and East Asian countries. It has been suggested that CagA proteins possessing an Eastern CM type are associated with less severe gastric disorders. In the present study, the effects of two CagA peptides with different CM motifs on cell function were compared: CagA with a Western and Eastern CM motif (CagA-WE), and CagA with two Western CM motifs (CagA-WW). CagA sequences were fused with green fluorescent protein (GFP) to form GFP-CagA fusion proteins. GFP-CagA and GFP control constructs were transfected into human gastric adenocarcinoma cells (AGS). GFP-CagA expression was verified by immunoblotting and immunofluorescence. The results demonstrated that, following 18 h, the CagA-WE-transfected cells were less adherent compared with the CagA-WW transfected cells. CagA has also been reported to cause cell elongation in AGS cells. In the current study, cell elongation was more frequent in the CagA-WW-transfected cells compared with the CagA-WE transfected cells (8.34 vs. 3.97% cells, respectively). The CagA peptides did not affect proliferation or apoptosis rates. These results suggest that different CM motif types may affect CagA virulence.

Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, long-term (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells.

Rab proteins are small GTPases required for vesicle trafficking through the secretory and endocytic pathways. Rab GDP-dissociation inhibitor (rab-GDI) regulates Rab protein function and localization by maintaining Rab proteins in the GDP-bound conformation. Two isoforms of rab-GDI are present in most mammalian cells: GDI-1 and GDI-2. It has recently been demonstrated that a Heat shock protein 90 (Hsp90) chaperone complex regulates the interactions between Rab proteins and Rab-GDI-1. The AR42J cell line is derived from rat pancreatic exocrine tumor cells and develops an acinar-like phenotype when treated with dexamethasone (Dex). The aim of the present study was to examine the expression of rab-GDI isoforms and Hsp90 in AR42J cells in the presence or absence of Dex. Rab-GDI: Hsp90 interactions were also examined. Both rab-GDI isoforms were detected in AR42J cells by immunoblotting. In Dex-treated cells, quantitative immunoblotting revealed that rab-GDI-1 expression increased by 28%, although this change was not statistically significant. Rab-GDI-2 levels were unaltered by Dex treatment. Approximately 21% rab-GDI-1 was membrane associated, whereas rab-GDI-2 was exclusively cytosolic. Dex treatment did not affect the subcellular distribution of rab-GDI isoforms. Hsp90 was present in the cytosolic and membrane fractions of AR42J cells and co-immunoprecipitated with cytosolic rab-GDI-1. Moreover, density gradient centrifugation of AR42J cell membranes revealed that Hsp90 and rab-GDI-1 co-localize on low- and high-density membrane fractions, including amylase-containing secretory granules. The Hsp90 inhibitor, geldanamycin, inhibited CCK-8-induced amylase release from these cells in a dose-dependent manner. Our results indicate that as AR42J cells differentiate into acinar-like cells, rab-GDI isoform expression and localization is not significantly altered. Moreover, our findings suggest that Hsp90 regulates agonist-induced secretion in exocrine cells by interacting with rab-GDI-1.