title: Identification of a signaling cascade for interleukin-8 production by Helicobacter pylori in human gastric epithelial cells.
abstract: Infecting gastric epithelial cells with Helicobacter pylori (H. pylori) has been shown to induce interleukin-8 (IL-8) production, but the signal transduction mechanism leading to IL-8 production is not defined clearly. In the present study, we investigated the molecular mechanism responsible for H. pylori-induced IL-8 release in human gastric epithelial cells. IL-8 levels in culture supernatants were determined by an enzyme linked-immunosorbent assay. Extracellular signal-regulated kinase (ERK) activity was tested using an in vitro kinase assay, which measured the incorporation of [gamma-33P]ATP into a synthetic peptide that is a specific ERK substrate. ERK phosphorylation and IkappaBalpha degradation by H. pylori infection were assessed by western blotting. In MKN45 cells, H. pylori-induced IL-8 release in a time-dependent manner. This IL-8 release was abolished by treatment with intracellular Ca2+ chelators (BAPTA-AM and TMB-8) but not by EGTA or nifedipine. The Ca2+ ionophore A23187 also induced IL-8 release to an extent similar to that of H. pylori infection. Calmodulin inhibitors (W7 and calmidazolium) and tyrosine kinase inhibitors (genistein and ST638) completely blocked IL-8 release by H. pylori and A23187. PD98059, an ERK pathway inhibitor, completely abolished H. pylori-induced IL-8 release. Moreover, BAPTA-AM, calmidazolium, and genistein, but not nifedipine, suppressed the ERK activation induced by H. pylori infection. PD98059 as well as MG132, an NF-kappaB pathway inhibitor, blocked both IL-8 production and degradation of IkappaBalpha induced by H. pylori infection, whereas only PD98059 inhibited ERK activity in response to H. pylori. There was no significant difference between IL-8 production induced by the cagA positive wild-type strain and the cagA negative isogenic mutant strain of H. pylori; therefore, CagA is not involved in the IL-8 production pathway. H. pylori-induced IL-8 production is dominantly regulated by Ca2+/calmodulin signaling, and ERK plays an important role in signal transmission for the efficient activation of H. pylori-induced NF-kappaB activity, resulting in IL-8 production.
authors.affiliation: Pharmacology Research Laboratory, Taiho Pharmaceutical Co., Ltd., 224-2 Ebisuno, Hiraishi, Kawauchi-cho, Tokushima, Japan. y-nozawa@taiho.co.jp
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Nozawa
authors.forename: Katsushi
authors.initials: K
authors.lastname: Nishihara
authors.forename: Richard M
authors.initials: RM
authors.lastname: Peek
authors.forename: Motoko
authors.initials: M
authors.lastname: Nakano
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Uji
authors.forename: Hirofusa
authors.initials: H
authors.lastname: Ajioka
authors.forename: Naosuke
authors.initials: N
authors.lastname: Matsuura
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Miyake
mesh_terms: D001422:Bacterial Adhesion / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D016480:Helicobacter pylori / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D016209:Interleukin-8 / Q000096:biosynthesis*; D007413:Intestinal Mucosa / Q000382:microbiology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D007375:Interleukin-1; D016209:Interleukin-8; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D011505:Protein-Tyrosine Kinases; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium