In-Cell Western analysis of Helicobacter pylori-induced phosphorylation of extracellular-signal related kinase via the transactivation of the epidermal growth factor receptor.
Helicobacter pylori activates extracellular-signal related (ERK) kinases in gastric epithelial cells, via transactivation of the EGF receptor (EGFR). H. pylori activation of EGFR may be relevant to epithelial hyperproliferation and gastric carcinogenesis. The aim of this study was to develop an 'In-Cell Western' (ICW) assay for quantitative examination of H. pylori-induced epithelial signalling, to enable the role of the EGFR in H. pylori-induced phosphorylation of ERK in epithelial cells to be ascertained. H. pylori strains were co-incubated with A431 and AGS cells. pERK and total ERK were quantified in situ using ICW analysis. H. pylori strains both with, and without a cag PAI, and Helicobacter felis, significantly increased pERK levels in A431 cells. The EGFR inhibitor EKB-569 dose-dependently reduced H. pylori-induced ERK phosphorylation in A431 and AGS cells. A significantly lower reduction was observed with cag+ strains in A431 but not AGS cells. The cag PAI was not necessary for EGFR signal transactivation. These data suggest that H. pylori induces pERK in epithelial cells partly via the EGFR pathway. Additional signalling mechanisms are likely to be involved in H. pylori-induced ERK phosphorylation. ICW analysis is a rapid quantitative method for evaluating the effects of inhibitors on H. pylori-induced cell signalling pathways of relevance to gastric carcinogenesis.
Du
Yiqi
Y
Leeds Institute of Molecular Medicine, St James's University Hospital, Leeds, LS9 7TF, UK.
Danjo
Kazuma
K
Robinson
Philip A
PA
Crabtree
Jean E
JE
D000634:Aminoquinolines; D000814:Aniline Compounds; D015153:Blotting, Western / Q000379:methods*; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000382:microbiology; D045744:Cell Line, Tumor; D004847:Epithelial Cells; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016481:Helicobacter Infections / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology; D016480:Helicobacter pylori / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System; D009930:Organic Chemicals / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000378:metabolism / Q000382:microbiology; D015533:Transcriptional Activation
D000634:Aminoquinolines; D000814:Aniline Compounds; D009930:Organic Chemicals; D047428:Protein Kinase Inhibitors; D066246:ErbB Receptors; D048049:Extracellular Signal-Regulated MAP Kinases; C413879:EKB 569