Helicobacter pylori induced transactivation of SRE and AP-1 through the ERK signalling pathway in gastric cancer cells.
OBJECTIVE
Helicobacter pylori infection induces expression of proinflammatory cytokines such as interleukin (IL)-8 and tumour necrosis factor alpha (TNF-alpha) in gastric mucosa, and their genes have AP-1 binding sites in the promoter region. c-Fos is important for transactivation of AP-1 which has SRE in the promoter region. We conducted this study to confirm H pylori induced transactivation of these binding sites.


METHODS
Transactivation of SRE and AP-1 was evaluated in human gastric cancer cells TMK1 and MKN45 by luciferase reporter assay in transient transfection. We compared the effects of coculture with four H pylori strains, a cag pathogenicity island (PAI) positive strain TN2, its isogenic vacA negative (TN2-DeltavacA) or cagE negative (TN2-DeltacagE) mutants, and a cag PAI negative clinical isolate T68. Phosphorylation of ERK1/2, JNK, and c-Jun was measured by immunoblot, induction of IL-8 secretion by ELISA, and the effects of MEK by inhibitor U0126.


RESULTS
Both SRE and AP-1 were transactivated by coculture with TN2. Although TN2-DeltavacA induced comparable transactivation, TN2-DeltacagE and T68 showed decreased transactivation of SRE (65% and 51%) and AP-1 (71% and 54%, respectively, of TN2). Heat killed TN2 or indirect contact using a permeable membrane inhibited transactivation. Levels of phosphorylated ERK1/2, JNK, and c-Jun were increased by coculture with TN2. MEK inhibitor U0126 reduced TN2 induced transactivation of SRE and AP1, as well as secretion of IL-8, by 83%, 87%, and 53%, respectively, of TN2.


CONCLUSIONS
Transactivation of SRE and AP-1, through ERK/MAPK and JNK/SAPK cascades, respectively, was found in gastric cancer cells cocultured with H pylori. Direct contact with viable bacteria possessing intact cag PAI is a prerequisite for the onset of intracellular signalling leading to AP-1 transactivation.
Mitsuno
Y
Y
Department of Gastroenterology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. MITSUNO-2IM@h.u-tokyo.ac.jp
Yoshida
H
H
Maeda
S
S
Ogura
K
K
Hirata
Y
Y
Kawabe
T
T
Shiratori
Y
Y
Omata
M
M
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002070:Butadienes / Q000494:pharmacology; D018899:CpG Islands / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005656:Fungal Proteins; D050980:GATA Transcription Factors; D016758:Genes, jun; D016480:Helicobacter pylori / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D016209:Interleukin-8 / Q000502:physiology; D020935:MAP Kinase Signaling System / Q000502:physiology*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000187:drug effects; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D013274:Stomach Neoplasms / Q000473:pathology / Q000503:physiopathology; D015533:Transcriptional Activation / Q000502:physiology*; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D002070:Butadienes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D050980:GATA Transcription Factors; D016209:Interleukin-8; D009570:Nitriles; C103937:PicB protein, Helicobacter pylori; C115209:Sre protein, Neurospora crassa; C113580:U 0126; C074689:VacA protein, Helicobacter pylori; D020929:Mitogen-Activated Protein Kinase Kinases