Helicobacter pylori-dependent ceramide production may mediate increased interleukin 8 expression in human gastric cancer cell lines.
OBJECTIVE
Helicobacter pylori adheres to gastric epithelial cells, activates nuclear factor kappaB (NF-kappaB), and stimulates interleukin (IL)-8 production, but the responsible molecular mechanisms remain largely unknown. Because several studies have shown that sphingolipids are involved in a number of signaling pathways, including NF-kappaB activation, we investigated the possible role of sphingolipids in the regulation of IL-8 expression in Kato III and AGS cells.


METHODS
IL-8 production in the conditioned media was quantified by enzyme immunoassay. Induction of messenger RNA (mRNA) was assessed by Northern blot analysis. Activation and binding activity of transcription factors were examined by luciferase assay and electrophoretic mobility shift assay, respectively. Intracellular levels of ceramide were quantified by diacylglycerol kinase assay.


RESULTS
A cell-permeable ceramide analogue (C2-ceramide) increased IL-8 expression with comparable mRNA induction. This effect was mimicked by sphingomyelinase, but not by phospholipase A2 or phospholipase C. C2-ceramide induced IL-8 gene transcription mainly through activation of NF-kappaB because mutation of the NF-kappaB-binding site completely abrogated the induction of luciferase activity. Direct contact of live H. pylori with epithelial cells increased the intracellular concentration of ceramide.


CONCLUSIONS
The results suggest a novel role of the sphingomyelin-ceramide pathway, at least in part through NF-kappaB, in IL-8 production induced by H. pylori infection in gastric epithelial cells.
Masamune
A
A
Third Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan. amasamune@int3.med.tohoku.ac.jp
Shimosegawa
T
T
Masamune
O
O
Mukaida
N
N
Koizumi
M
M
Toyota
T
T
D002518:Ceramides / Q000096:biosynthesis* / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016481:Helicobacter Infections / Q000378:metabolism*; D016480:Helicobacter pylori; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D013274:Stomach Neoplasms / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology; D014407:Tumor Cells, Cultured
D002518:Ceramides; D004791:Enzyme Inhibitors; D016209:Interleukin-8; C064769:N-acetylsphingosine; D016328:NF-kappa B; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D013108:Sphingomyelin Phosphodiesterase; D013110:Sphingosine