Abstract
The Helicobacter pylori CagA protein is translocated into gastric epithelial cells through a type IV secretion system (TFSS), and published studies suggest CagA is critical for H. pylori-associated carcinogenesis. CagA is thought to be necessary and sufficient to induce the motogenic response observed in response to CagA+ strains, as CagA interacts with proteins involved in adhesion and motility. We report that H. pylori strain 60190 stimulated AGS cell motility through a CagA- and TFSS-dependent mechanism, because strains 60190DeltacagA or 60190DeltacagE (TFSS-defective) did not increase motility. The JNK pathway is critical for H. pylori-dependent cell motility, as inhibition using SP600125 (JNK1/2/3 inhibitor) or a JNK2/3-specific inhibitor blocked motility. JNK mediates H. pylori-induced cell motility by activating paxillin, because JNK inhibition blocked paxillinTyr-118 phosphorylation, and paxillin expression knockdown completely abrogated bacteria-induced motility. Furthermore, JNK and paxillinTyr-118 were activated by 60190DeltacagA but not 60190DeltacagE, demonstrating CagA-independent signaling critical for cell motility. A beta1 integrin-blocking antibody significantly inhibited JNK and paxillinTyr-118 phosphorylation and cell scattering, demonstrating that CagA-independent signaling required for cell motility occurs through beta1. The requirement of both Src and focal adhesion kinase for signaling and motility further suggests the importance of integrin signaling in H. pylori-induced cell motility. Finally, we show that JNK activation occurs independent of known upstream kinases and signaling molecules, including Nod1, Cdc42, Rac1, MKK4, and MKK7, which demonstrates novel signaling leading to JNK activation. We report for the first time that H. pylori mediates CagA-independent signaling that promotes cell motility through the beta1 integrin pathway.
Highlights
Helicobacter pylori infects one-half of the world’s population, establishing a chronic infection in the gastric mucosa that persists for the lifetime of the host [1, 2]
We identified paxillin as a downstream target of H. pylori-dependent JNK activation and that paxillin activity is required for H. pylori-stimulated gastric cancer cell motility
These data show that JNK signaling, which is required for H. pylori-stimulated cell motility, is activated in a CagA-independent but still TFSS-dependent manner
Summary
Cell Culture and Reagents—AGS gastric adenocarcinoma cells (ATCC, Manassas, VA) were cultured in Ham’s F-12 media (Mediatech, Herndon, VA) supplemented with 10% fetal calf serum (Gemini, West Sacramento, CA), 100 g/ml streptomycin, 100 IU/ml penicillin (Mediatech, Herndon, VA), and 0.0375% (w/v) sodium bicarbonate (Mediatech, Herndon, VA) at 37 °C in 5% CO2. 1.25 ϫ 104 AGS cells were seeded into a 24-well plate, and the following day were transfected with Lipofectamine 2000 (Invitrogen) according to product protocol using 1.5 l of stock Cdc or luciferase siRNA (20 M; a kind gift from Dr Marc Symons, Feinstein Institute for Medical Research, Manhasset, NY). After 2 days fresh medium was added, and cells were either infected with H. pylori for scatter assays or collected for Western blot analysis. The following day, cells were infected with H. pylori strains for either 1 h (Western blot analysis) or overnight. Western blot analysis was first performed on whole bacteria lysates to confirm that the mutant strains 60190⌬cagE and 60190⌬vacA, but not 60190⌬cagA, expressed CagA (Fig. 1B). Pylori plays no role in the induction of this morphological response
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