cDNA Microarray Analysis of Helicobacter pylori-Mediated Alteration of Gene Expression in Gastric Cancer Cells

doi:10.1006/bbrc.2001.5006

Biochemical and Biophysical Research Communications

Volume 284, Issue 2, 8 June 2001, Pages 443-449

https://doi.org/10.1006/bbrc.2001.5006 Get rights and content

Abstract

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, IκBα, 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 (ΔcagE), 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-κB activation, indicating that H. pylori-mediated A20 expression could be a negative regulator of NF-κB 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.

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A20 (also known as TNFAIP3) is a cytoplasmic protein that plays a key role in the negative regulation of inflammation and immunity. Polymorphisms in the A20 gene locus have been identified as risk alleles for multiple human autoimmune diseases, and A20 has also been proposed to function as a tumor suppressor in several human B-cell lymphomas. A20 expression is strongly induced by multiple stimuli, including the proinflammatory cytokines TNF and IL-1, and microbial products that trigger pathogen recognition receptors, such as Toll-like receptors. A20 functions in a negative feedback loop, which mediates its inhibitory functions by downregulating key proinflammatory signaling pathways, including those controlling NF-κB- and IRF3-dependent gene expression. Activation of these transcription factors is controlled by both K48- and K63- polyubiquitination of upstream signaling proteins, respectively triggering proteasome-mediated degradation or interaction with other signaling proteins. A20 turns off NF-κB and IRF3 activation by modulating both types of ubiquitination. Induction of K48-polyubiquitination by A20 involves its C-terminal zinc-finger ubiquitin-binding domain, which may promote interaction with E3 ligases, such as Itch and RNF11 that are involved in mediating A20 inhibitory functions. A20 is thought to promote de-ubiquitination of K63-polyubiquitin chains either directly, due to its N-terminal deubiquitinase domain, or by disrupting the interaction between E3 and E2 enzymes that catalyze K63-polyubiquitination. A20 is subject to different mechanisms of regulation, including phosphorylation, proteolytic processing, and association with ubiquitin binding proteins. Here we review the expression and biological activities of A20, as well as the underlying molecular mechanisms.
Pathogenomics of Helicobacter
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The pathogenic bacterium Helicobacter pylori infects half of the human population and is one of the genetically most diverse bacterial species known. H. pylori was one of the first bacterial species whose genome was sequenced in 1997, and the first species for which two complete sequences from independent isolates were available for within-species comparisons. For almost 10 years, genomic and post-genomic analysis has contributed enormously to our understanding of the pathogenesis of H. pylori infection. This review summarizes the available information, emphasizing work performed in the framework of the PathoGenoMik funding initiative (2001–2006) of the German Ministry of Education and Research.
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Global analysis of gene expression profiles in stomach samples from human beings and rhesus macaques identified large sets of inflammatory and immune response genes up-regulated during H pylori infection.27,28 This proinflammatory gene expression signature in stomach samples is much larger than that observed using gastric epithelial cell lines infected with H pylori in vitro.8,26 In epithelia, gene expression changes are largely dependent on t4ss-mediated translocation of bacterial macromolecules into the cells.8,10
Background & Aims: Recognition of infection leads to induction of adaptive immunity through activation of antigen-presenting cells (APCs). Among APCs, dendritic cells (DCs) have the unique capacity to deliver antigens from the periphery to T cells in secondary lymphoid organs. Methods: We analyzed molecular mechanisms of the Helicobacter pylori–induced APC activation in vitro and investigated the influence of Myd88 signaling on the phenotype of adaptive immunity to H pylori in a murine infection model. Results: The adaptor protein Myd88 mediates Toll-like receptor (TLR), interleukin (IL)-1, and IL-18 signaling. DCs from wild-type, IL-1R^−/−, and IL-18^−/− mice responded to H pylori with secretion of proinflammatory cytokines and up-regulation of major histocompatibility complex II and costimulatory molecules. In Myd88^−/− DCs these processes were impaired profoundly, showing that TLR-dependent H pylori–sensing affects DC activation. Analysis of the H pylori–specific DC transcriptome revealed that large parts of the bacteria-induced transcriptional changes depended on Myd88 signaling, comprising numerous genes involved in crucial steps of immune regulation, such as DC maturation/differentiation, antigen uptake/presentation, and effector cell recruitment/activation. The impaired ability of Myd88^−/− DCs, B cells, and macrophages to mount a proinflammatory response to H pylori in vitro was reflected in vivo by reduced gastric inflammation and increased bacterial colonization in Myd88-deficient mice. Furthermore, Helicobacter-specific IgG2c/IgG1 ratios were reduced in Myd88^−/− animals, suggesting the involvement of the Myd88-dependent pathway in the instruction of adaptive immunity toward a T helper cell type 1 phenotype. Conclusions: A principal pathway by which DCs sense H pylori and become activated is the TLR-dependent signaling cascade. In vivo, Myd88 signaling affects adaptive immunity to the bacterium.
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2006, Molecular and Cellular Proteomics
Helicobacter pylori infection is a crucial factor in the pathogenesis of several digestive disorders, including peptic ulcers, chronic gastritis, and gastric cancer. Moreover H. pylori induces disease-specific protein expression in gastric epithelial cells. The aim of the present study was to characterize proteins differentially expressed in H. pylori-infected gastric epithelial AGS cells. An in vitro model was established using a multiplicity of infection of 100 and evaluating the effectiveness of H. pylori infection by functional analyses. Changes in protein patterns were identified using a proteomic approach consisting of two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. The expression of many proteins was found to be altered, and 28 of these were identified and classified as protein synthesis- and folding-related proteins, cytoskeleton proteins, metabolic enzymes, transcription- and translation-related proteins, angiogenesis/metastasis-related proteins, cell communication/signal transduction-related proteins, or others (oxygen-regulated protein and oncoprotein). The expression profiles of eight of these proteins, laminin γ-1 chain precursor, valosin-containing protein, heat shock 70-kDa protein, mitochondrial matrix protein P1, FK506-binding protein 4, T-complex protein 1, enolase α, and 14-3-3 β were further examined in cancerous and paired surrounding normal tissues by immunoblot assay and immunohistochemical staining to identify molecular targets that may be involved in the pathogenesis of H. pylori-induced gastric diseases. On the basis of our results, valosin-containing protein, mitochondrial matrix protein P1, T-complex protein 1, enolase α, and 14-3-3 β may play a crucial role in H. pylori-induced gastric carcinogenesis by mediating antiapoptotic and proliferative responses.

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^☆: Abbreviations used: PAI, pathogenicity island; TNF, tumor necrosis factor; IL, interleukin.

¹: To whom correspondence and reprint requests should be addressed. Fax: 3-3814-0021. E-mail: [email protected].

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Regular ArticlecDNA Microarray Analysis of Helicobacter pylori-Mediated Alteration of Gene Expression in Gastric Cancer Cells☆

Abstract

Gastroenterology

J. Biol. Chem.

Gastroenterology

Gastroenterology

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Cell

Effect of treatment of Helicobacter pylori infection on the long-term recurrence of gastric or duodenal ulcer: A randomized, controlled study

Ann. Intern. Med.

Helicobacter pylori infection and the risk of gastric carcinoma

N. Engl. J. Med.

Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii

N. Engl. J. Med.

Activation of IL-8 gene expression by Helicobacter pylori is regulated by transcription factor nuclear factor-kappa B in gastric epithelial cells

J. Immunol.

Mechanisms involved in Helicobacter pylori-induced interleukin 8 production by gastric cancer cell line, MKN45

Infect. Immun.

Interleukin-8 expression in Helicobacter pylori infected, normal, and neoplastic gastroduodenal mucosa

J. Clin. Pathol.

Heightened inflammatory response and cytokine expression in vivo to cagA+ Helicobacter pylori strains

Lab. Invest.

cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors

Proc. Natl. Acad. Sci. USA

Structure of cag pathogenicity island in Japanese Helicobacter pylori isolates

Gut

Regular Article
cDNA Microarray Analysis of Helicobacter pylori-Mediated Alteration of Gene Expression in Gastric Cancer Cells☆