CagA protein secreted by the intact type IV secretion system leads to gastric epithelial inflammation in the Mongolian gerbil model.

Helicobacter pylori causes various gastro-duodenal diseases, including gastric cancer. The CagA protein, an H. pylori virulence factor, induces morphological changes in host cells and may be associated with the development of peptic ulcer and gastric carcinoma. The present study has analysed the role of CagA protein in the pathogenesis of H. pylori infection in the Mongolian gerbil model. Mongolian gerbils were challenged with wild-type H. pylori strain TN2, which has a functional cag pathogenicity island or isogenic mutants with disrupted cagA (DeltacagA) or cagE (DeltacagE) genes. They were sacrificed at 7, 13, and 25 weeks after inoculation. Pathological changes of the gastric mucosa were determined and apoptosis was assessed by the TUNEL assay. Immunohistochemistry for PCNA, phospho-IkappaBalpha, and phospho-Erk was also performed. All of the bacterial strains colonized the gerbil stomach at similar densities; however, the DeltacagA mutant induced milder gastritis than did the wild type. The extent of apoptosis and lymphoid follicle formation in the epithelium appeared to depend on intact cagA. The DeltacagA mutant induced less phosphorylation of IkappaBalpha and Erk, and less expression of interferon-gamma and interleukin-1beta mRNA in the epithelium than did the wild type. It is concluded that CagA protein may be essential for the induction of severe gastritis in the Mongolian gerbil model.

Analysis of apoptotic and antiapoptotic signalling pathways induced by Helicobacter pylori.

BACKGROUND AND AIMS: Although it is reported that Helicobacter pylori induces apoptosis on gastric epithelial cells, the mechanism remains unknown. Antiapoptotic effects generated by H pylori have not yet been evaluated. METHODS: (1) H pylori strains (type 1 wild, TN2-deltacagE, TN2-deltavacA) were cocultured with MKN45, TMK1, and HeLa cells, and cell viability and apoptosis were assessed by trypan blue exclusion and DNA laddering, respectively. (2) Activation of caspases-3, 7, and 8, cytochrome c release from the mitochondria, and Fas, Fas associated death domain protein (FADD), Bax, Bak, and Bcl-X expression were evaluated by immunoblot analysis. (3) To investigate whether nuclear factor kappa B (NFkappaB) activation induced by cag pathogenicity island (PAI) positive H pylori affects antiapoptosis, MKN45 cells stably expressing super-repressor IkappaBalpha were cocultured with H pylori, and cell viability and caspase activation were evaluated. NFkappaB regulated gene expression was also evaluated by ribonuclease protection assay. RESULTS: (1) Wild-type and deltavacA mutant H pylori induced apoptosis more potently than the deltacagE mutant. Inhibition of cell contact between H pylori and cancer cells and heat killing H pylori diminished cell death. (2) Caspases-3, 7, and 8 were activated time dependently by H pylori as well as by the agonist anti-Fas. Cytochrome c release from mitochondria was observed and was not inhibited by caspase-8 inhibitor. Although protein expression of Fas, FADD, Bax, Bak, and Bcl-X in the whole cell lysates was not changed by H pylori, Bax was decreased from mitochondria free cytosol suggesting that Bax was translocated into mitochondria. (3) Cell death and the activities of caspases-3 and 8 were promoted in MKN45 cells stably expressing super-repressor IkappaBalpha that inhibits NFkappaB activation. Antiapoptotic proteins c-IAP1 and c-IAP2 were upregulated by the wild-type strains. CONCLUSION: cag PAI positive H pylori is capable of inducing apoptotic effects mainly through the mitochondrial pathway. Antiapoptotic effects mediated by NFkappaB activation were also observed.

Analysis of apoptotic and antiapoptotic signalling pathways induced by Helicobacter pylori.

BACKGROUND AND AIMS: Although it is reported that Helicobacter pylori induces apoptosis on gastric epithelial cells, the mechanism remains unknown. Antiapoptotic effects generated by H pylori have not yet been evaluated. METHODS: (1) H pylori strains (type 1 wild, TN2-DeltacagE, TN2-DeltavacA) were cocultured with MKN45, TMK1, and HeLa cells, and cell viability and apoptosis were assessed by trypan blue exclusion and DNA laddering, respectively. (2) Activation of caspases-3, 7, and 8, cytochrome c release from the mitochondria, and Fas, Fas associated death domain protein (FADD), Bax, Bak, and Bcl-X expression were evaluated by immunoblot analysis. (3) To investigate whether nuclear factor kappa B (NFkappaB) activation induced by cag pathogenicity island (PAI) positive H pylori affects antiapoptosis, MKN45 cells stably expressing super-repressor Ikappabetaalpha were cocultured with H pylori, and cell viability and caspase activation were evaluated. NFkappaB regulated gene expression was also evaluated by ribonuclease protection assay. RESULTS: (1) Wild-type and DeltavacA mutant H pylori induced apoptosis more potently than the DeltacagE mutant. Inhibition of cell contact between H pylori and cancer cells and heat killing H pylori diminished cell death. (2) Caspases-3, 7, and 8 were activated time dependently by H pylori as well as by the agonist anti-Fas. Cytochrome c release from mitochondria was observed and was not inhibited by caspase-8 inhibitor. Although protein expression of Fas, FADD, Bax, Bak, and Bcl-X in the whole cell lysates was not changed by H pylori, Bax was decreased from mitochondria free cytosol suggesting that Bax was translocated into mitochondria. (3) Cell death and the activities of caspases-3 and 8 were promoted in MKN45 cells stably expressing super-repressor Ikappabetaalpha that inhibits NFkappaB activation. Antiapoptotic proteins c-IAP1 and c-IAP2 were upregulated by the wild-type strains. CONCLUSION: cag PAI positive H pylori is capable of inducing apoptotic effects mainly through the mitochondrial pathway. Antiapoptotic effects mediated by NFkappaB activation were also observed.

Systematic analysis of the TGF-beta-Smad signaling pathway in gastrointestinal cancer cells.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway has an important role in carcinogenesis. To study the frequency and mechanism of functional impairment of this pathway in human gastrointestinal cancers, we used a reporter assay to examine the response of 38 cell lines (11 colorectal, 9 pancreatic, 10 gastric, and 8 hepatic cancers) to TGF-beta. We then analyzed TGF-beta type II receptor (T beta RII) gene, immunoblots of Smad4, and restoration of the pathway by rescuing T beta R or Smad. We observed impaired signaling in 91% of colorectal, 67% of pancreatic, and 40% of gastric cancer cell lines, but in none of the hepatic cancer cells. We suggest that this pathway does not function as a tumor suppressor in hepatic carcinogenesis. The impairment is due to inactivation of T beta RII and Smad4 in colorectal and pancreatic cancers. However, because the signal was not recovered by rescuing T beta R or Smad genes in TGF-beta-response-defective gastric cancer cell lines, we suggest that novel molecules or mechanisms are involved in the impaired pathway in some gastric cancers.

Distinct mechanism of Helicobacter pylori-mediated NF-kappa B activation between gastric cancer cells and monocytic cells.

NF-kappaB is a critical regulator of genes involved in inflammation. Gastric epithelial cells and macrophages are considered the main sources of pro-inflammatory cytokines. We investigated NF-kappaB activation by Helicobacter pylori in MKN45 gastric epithelial cells and THP-1 monocytic cells. Although, cag pathogenicity island (PAI)-positive H. pylori (wild type) activated NF-kappaB in both cells, isogenic mutant of cagE (DeltacagE) activated it only in THP-1 cells. Supernatant from the wild type culture could activate NF-kappaB in THP-1 cells but not in MKN45 cells. High density cDNA array analysis revealed that mRNA expression of NF-kappaB-regulated genes such as interleukin (IL)-8, tumor necrosis factor-alpha (TNFalpha), and IL-1beta was significantly up-regulated by the wild type in both cells, whereas it was up-regulated by DeltacagE only in THP-1 cells. Experiments using CD14-neutralizing antibody and IL-1 receptor-associated kinase (IRAK) assay showed that both wild type and DeltacagE H. pylori activated NF-kappaB through CD14 and IRAK in THP-1 cells but not in MKN45 cells. Macrophages from C3H/HeJ mice carrying point mutation in the Toll-like receptor 4 (TLR4) gene showed decreased NF-kappaB activation and TNFalpha secretion compared with C3H/HeN mouse macrophage when treated with H. pylori. In conclusion, H. pylori-induced NF-kappaB activation in epithelial cells is dependent on cag PAI and contact but does not involve CD14 and IRAK, whereas in macrophage/monocytic cells it is independent of cag PAI or contact but involves CD14 and TLR4.

Helicobacter pylori induced transactivation of SRE and AP-1 through the ERK signalling pathway in gastric cancer cells.

BACKGROUND AND AIMS: 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.

cDNA microarray analysis of Helicobacter pylori-mediated alteration of gene expression in gastric cancer cells.

Helicobacter pylori infection stimulates several intracellular signaling pathways and is accompanied by increased gene expression in gastric epithelial cells. High-density cDNA microarray was used to characterize the mRNA expression profile of genes in human gastric cancer cells (MKN45, AGS) cocultured with H. pylori. Coculture with cag pathogenicity island (PAI)-positive H. pylori (wild-type) significantly up-regulated mRNA expression in 8 of 2304 genes tested. In 6 (interleukin-8, I(kappaB)alpha, A20, ERF-1, keratin K7, glutathione peroxidase) of the 8 genes, up-regulation was confirmed by RT-PCR. In coculture with isogenic cagE-negative mutant ((Delta)cagE), which encodes a type IV secretion system with other genes in the cag PAI, no significant up-regulation was found. We further analyzed the role of A20. Transfection of expression vector encoding A20 resulted in an inhibition of H. pylori-mediated NF-kappaB activation, indicating that H. pylori-mediated A20 expression could be a negative regulator of NF-kappaB activation. Taken together, these results indicate the importance of microarray technology as a tool for analyzing the complex interplay between H. pylori and the host.

Helicobacter pylori activates the cyclin D1 gene through mitogen-activated protein kinase pathway in gastric cancer cells.

Helicobacter pylori induces cellular proliferation in host cells, but the mechanism remains unclear. Thus, we examined the effect of H. pylori on cyclin D1, an important regulator of the cell cycle, especially in relation to intracellular signaling pathways. In a Northern blot analysis, cyclin D1 transcription in gastric cancer (AGS) cells was enhanced by coculture with H. pylori strain TN2 in a time-dependent and multiplicity-of-infection-dependent manner. An isogenic mutant form of vacA also increased cyclin D1 transcription, but mutant forms of cagE or the entire cag pathogenicity island did not enhance cyclin D1 transcription. These effects were confirmed with a luciferase assay of the cyclin D1 promoter (pD1luc). Cyclin D1 promoter activation by H. pylori was inhibited by MEK inhibitors (U0126 and PD98059), indicating that the mitogen-activated protein kinase pathway may be involved in intracellular signal transduction. In contrast, transfection of a reporter plasmid having any point mutations of the NF-kappaB binding sites in the promoter (pD1-kappaB1M, pD1-kappaB2M, or pD1-kappaB1/2M) or cotransfection of dominant negative IkappaBalpha did not affect cyclin D1 activation by H. pylori. In conclusion, H. pylori activates cyclin D1 through the mitogen-activated protein kinase pathway and not through NF-kappaB activation in AGS cells. This activation of cyclin D1 is partly dependent on the cag pathogenicity island but not on vacA.

Determination of Helicobacter pylori virulence by simple gene analysis of the cag pathogenicity island.

Nucleic acid amplification was performed for five loci in the cag pathogenicity island (PAI) of Helicobacter pylori (comprising cagA, the cagA promoter region, cagE, cagT, and the left end of cagII [LEC]), and gastric inflammation in patients was evaluated. Of 204 H. pylori isolates from Japanese patients (53 with peptic ulcer, 55 with gastric cancer, and 96 with chronic gastritis), 197 (96.6%) were positive for all five loci. Two isolates (1%) were negative for all five loci, and five isolates (2.4%) were positive for only cagA and LEC. These latter seven isolates were all from patients with mild chronic gastritis. Neutrophil infiltration in gastric mucosa was significantly milder in patients infected with partially or totally deleted-PAI strains than in those with intact-PAI strains. The cagE gene was a more accurate marker of an intact cag PAI than the cagA gene, and cagE seemed to be more useful in discriminating between H. pylori strains causing different rates of disease progression.

H. pylori activates NF-kappaB through a signaling pathway involving IkappaB kinases, NF-kappaB-inducing kinase, TRAF2, and TRAF6 in gastric cancer cells.

BACKGROUND & AIMS: H. pylori infection on gastric epithelial cells has been shown to induce NF-kappaB activation, but the mechanism of intracellular signal conduction that leads to NF-kappaB activation is not clear. The aim of this study was to analyze the molecular mechanism responsible for H. pylori-mediated NF-kappaB activation on gastric cancer cells. METHODS: NF-kappaB activation by H. pylori was tested by using luciferase reporter assay. IkappaBalpha degradation by H. pylori infection was assessed by immunoblotting. IKKalpha and IKKbeta activation was analyzed by kinase assay. In transfection experiments, effects of dominant negative IkappaBalpha, IKKalpha, IKKbeta, NF-kappaB-inducing kinase (NIK), TRAF2, and TRAF6 mutants were investigated. The effects of an IKKbeta-specific inhibitor, aspirin, on NF-kappaB activation and IL-8 secretion were also analyzed. RESULTS: H. pylori promotes degradation of IkappaBalpha, a cytoplasmic inhibitor of NF-kappaB. In kinase assay, H. pylori induced IKKalpha and IKKbeta catalytic activity in gastric cancer cells. Transfection of kinase-deficient mutant of either IKK inhibited H. pylori-mediated NF-kappaB activation dose-dependently. Aspirin inhibited both NF-kappaB activation and IL-8 secretion induced by H. pylori. NF-kappaB activation was also inhibited by transfection of kinase-deficient NIK or a dominant negative mutant of upstream adapter protein TRAF2 or TRAF6. CONCLUSIONS: H. pylori induces NF-kappaB activation through an intracellular signaling pathway that involves IKKalpha, IKKbeta, NIK, TRAF2, and TRAF6.