Mechanisms involved in Helicobacter pylori-induced interleukin-8 production by a gastric cancer cell line, MKN45.
Accumulating evidence suggests an important role of interleukin-8 (IL-8) in Helicobacter pylori infection-associated chronic atrophic gastritis and peptic ulcer. We observed in this study that a gastric cancer-derived cell line, MKN45, produced a massive amount of IL-8 upon coculture with live H. pylori but not with killed H. pylori, H. pylori culture supernatants, or live H. pylori separated by a permeable membrane, indicating that IL-8 production requires a direct contact between the cells and live bacteria. Moreover, the tyrosine kinase inhibitor herbimycin but neither a protein kinase C inhibitor (staurosporine) nor a protein kinase A inhibitor (H89) inhibited IL-8 production by MKN45 cells cocultured with live bacteria, suggesting the involvement of a tyrosine kinase(s) in H. pylori-induced IL-8 production. In addition, coculture of H. pylori induced IL-8 mRNA expression in MKN45 cells and an increase in luciferase activity in cells which were transfected with a luciferase expression vector linked with a 5'-flanking region of the IL-8 gene (bp -133 to +44), indicating that the induction of IL-8 production occurred at the transcriptional level. This region contain three cis elements important for induction of IL-8 gene expression: AP-1 (-126 to -120 bp), NF-IL6 (-94 to -81 bp), and NF-kappaB (-80 to -70 bp) binding sites. Mutation of the NF-kappaB binding site abrogated completely the induction of luciferase activity, whereas that of the AP-1 site partially reduced the induction. However, mutation of the NF-IL6 binding site resulted in no decrease in the induction of luciferase activity. Moreover, specific NF-kappaB complexes were detected in the nuclear proteins extracted from MKN45 cells which were infected with H. pylori. Collectively, these results suggest that H. pylori induced the activation of NF-kappaB as well as AP-1, leading to IL-8 gene transcription.
Aihara
M
M
Microbiological Research Institute, Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan. m_aihara@research.otsuka.co.jp
Tsuchimoto
D
D
Takizawa
H
H
Azuma
A
A
Wakebe
H
H
Ohmoto
Y
Y
Imagawa
K
K
Kikuchi
M
M
Mukaida
N
N
Matsushima
K
K
D005786:Gene Expression Regulation; D016480:Helicobacter pylori / Q000502:physiology*; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D013274:Stomach Neoplasms / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D016209:Interleukin-8; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D011505:Protein-Tyrosine Kinases