Abstract
The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The formed vacuole is assumed to be a hybrid of late endosome and lysosome. To elucidate the molecular mechanism of VacA-induced vacuolation, we examined the participation of syntaxin 7 in the human gastric epithelial cell line AGS. Immunocytochemistry revealed that endogenous syntaxin 7 was localized to vacuoles induced by VacA. Northern and Western blotting demonstrated that VacA intoxication increased syntaxin 7 mRNA and protein expression, respectively, in a time-dependent manner. Transient transfection of dominant-negative mutant syntaxin 7, which lacks a carboxyl-terminal transmembrane domain, inhibited VacA-induced vacuolation. In contrast, transient transfection of wild-type syntaxin 7, dominant-negative mutant syntaxin 1a, or dominant-negative mutant syntaxin 4 did not alter VacA-induced vacuolation. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with mutant syntaxin 7 but not in cells stably transfected with wild-type syntaxin 7, mutant syntaxin 1a, or mutant syntaxin 4. Sequential immunocytochemical observation confirmed that expression of mutant syntaxin 7 did not affect VacA attachment to or internalization into AGS cells. We suggest that syntaxin 7 is involved in the intracellular vacuolation induced by VacA.
Highlights
Tragastric administration of VacA to mice induced erosion of gastric epithelium [1]
Localization of Endogenous Syntaxin 7 to VacA-induced Vacuoles in AGS Cells—In the first attempt to investigate whether syntaxin 7 participates in the VacA-induced vacuolization in AGS cells, we examined the localization of endogenous syntaxin 7 in VacA-treated and untreated AGS cells by immunocytochemistry
VacA Increased the Expression of Syntaxin 7 mRNA and Protein in AGS Cells—We examined the expression of endogenous syntaxin 7 in VacA-treated AGS cells by both Northern and Western blotting
Summary
Tragastric administration of VacA to mice induced erosion of gastric epithelium [1]. In vitro, VacA induces cytoplasmic vacuolation. The intracellular vacuolation is assumed to represent an early pathophysiological event leading to cell sufferance and eventually to necrosis These vacuoles induced by VacA contain both a late endosomal marker, rab, and a lysosomal marker, lgp110, suggesting that they are hybrids of late endosomes and lysosomes [2, 3]. We reported that dynamin, a large molecular weight GTP-binding protein functioning as a mechanochemical enzyme in vesicle formation, is involved in VacA-induced vacuolation [5]. A novel member of syntaxin family, syntaxin 7, was identified and characterized It was localized on both late endosome and lysosome and was elucidated to play a crucial role in the physiological heterotypic fusion of late endosome and lysosome [12, 13]. We concluded that syntaxin 7 is involved in the molecular mechanism of VacA-induced vacuolation
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