title: Helicobacter pylori vacuolating cytotoxin induces activation of the proapoptotic proteins Bax and Bak, leading to cytochrome c release and cell death, independent of vacuolation.
abstract: Helicobacter pylori vacuolating cytotoxin, VacA, which causes vacuolation of gastric epithelial cells and other types of cultured cells, is known to stimulate apoptosis via a mitochondria-dependent pathway. In the present study, we examined the mechanisms of VacA-induced mitochondrial damage. Intracellular VacA localization was monitored by immunostaining and confocal microscopy; in AZ-521 cells in which cytochrome c release was stimulated, most of VacA was localized to vacuoles rather than mitochondria. VacA reduced the membrane potential of isolated mitochondria without inducing cytochrome c release, suggesting that it did not act directly to induce cytochrome c release from mitochondria and that in intact cells, VacA-induced cytochrome c release involved apoptosis-related factor(s), such as a proapoptotic Bcl-2 family protein. In agreement, flow cyto-metric analyses using antibodies specific for activated Bax revealed that intracellular Bax was activated by VacA in a concentration- and time-dependent manner. Using active form-specific antibodies, we also observed that the Bcl-2 family protein, Bak, was activated. By confocal microscopy, Bax and Bak were activated in AZ-521 cells in which cyto-chrome c release was induced by VacA. In addition, small interfering RNA-induced silencing of the bax gene resulted in reduction of VacA-stimulated cytochrome c release, consistent with a contribution of VacA-induced Bax activation to cytochrome c release. NH4Cl enhanced both VacA-induced vacuolation and Bax activation, whereas Bax activation was not inhibited by bafilomycin A1, which inhibited vacuolation caused by VacA. These results suggest that VacA acts through different signaling pathways to induce apoptosis via Bax activation, independent of vacuolation.
authors.affiliation: PRESTO, Japan Science and Technology Corporation, Saitama 332-0012, Japan.
authors.forename: Eiki
authors.initials: E
authors.lastname: Yamasaki
authors.forename: Akihiro
authors.initials: A
authors.lastname: Wada
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kumatori
authors.forename: Ichiro
authors.initials: I
authors.lastname: Nakagawa
authors.forename: Junko
authors.initials: J
authors.lastname: Funao
authors.forename: Masaaki
authors.initials: M
authors.lastname: Nakayama
authors.forename: Junzo
authors.initials: J
authors.lastname: Hisatsune
authors.forename: Miyuki
authors.initials: M
authors.lastname: Kimura
authors.forename: Joel
authors.initials: J
authors.lastname: Moss
authors.forename: Toshiya
authors.initials: T
authors.lastname: Hirayama
mesh_terms: D017209:Apoptosis; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D016923:Cell Death; D002460:Cell Line; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism*; D005434:Flow Cytometry; D006367:HeLa Cells; D016480:Helicobacter pylori / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D018942:Macrolides / Q000494:pharmacology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D011522:Protons; D034741:RNA, Small Interfering / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014617:Vacuoles / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D018942:Macrolides; D011522:Protons; D034741:RNA, Small Interfering; C074689:VacA protein, Helicobacter pylori; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; C040929:bafilomycin A1; D045304:Cytochromes c