Conditional gene silencing utilizing the lac repressor reveals a role of SHP‐2 in cagA‐positive Helicobacter pylori pathogenicity

Higuchi, M; Tsutsumi, Ryouhei; Higashi, Hideaki; Hatakeyama, Masanori

doi:10.1111/j.1349-7006.2004.tb03229.x

Cited by 54 publications

(47 citation statements)

References 30 publications

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“…Higashi et al [22] have also found that ERK is activated by CagA via SHP-2 recruitment and activation in AGS cells. Similarly, CagA expression in gastric epithelial cells results in sustained ERK MAP kinase activation [23]. The research of Zhu et al [24] further confirms that the transformation of immortalized gastric epithelial cells is induced by CagA via the ERK/MAPK pathway.…”

Section: Discussionsupporting

confidence: 55%

Human gastrin mRNA expression up-regulated by Helicobacter pylori CagA through MEK/ERK and JAK2-signaling pathways in gastric cancer cells

et al. 2011

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Background Helicobacter pylori-cytotoxin-associated protein A (CagA) and gastrin are believed to play an important role in gastric carcinogenesis, but their interaction has been incompletely clear. Methods We constructed a eukaryotic expression vector pcDNA3.1/cagA and a luciferase reporter vector pGL/ gastrin promoter, and then co-transfected them into gastric cancer AGS and SGC-7901 cells. The two kinds of gastric cancer cells were, respectively, infected with cagA-positive H. pylori NCTC11637, and then the gastrin promoter activity and gastrin mRNA level were detected with a Dual-Luciferase reporter assay system and quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively. Next, after the MEK/ERK and JAK2-signaling pathway inhibitors, U0126 and AG490, were used to treat the two cell lines, or the ERK1 and JAK2 genes were knocked down by siRNAs (small interference RNAs) in the two cell lines, the gastrin promoter activity and gastrin mRNA level were observed again. Results The results indicated that CagA could activate the gastrin promoter and up-regulate gastrin mRNA expression in AGS and SGC-7901 cells, but these effects could be inhibited by the inhibitors U0126 and AG490, and the CagAinduced gastrin mRNA expression was down-regulated in the cells whose ERK1 or JAK2 gene was knocked down.

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Section: Discussionsupporting

confidence: 55%

Human gastrin mRNA expression up-regulated by Helicobacter pylori CagA through MEK/ERK and JAK2-signaling pathways in gastric cancer cells

et al. 2011

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“…In this study, we used a pair of isogenic strains (147A and 147C) with identical CagA proteins, differing only by the presence of a single C-EPIYA motif (31)(32)(33) to elucidate the mechanisms by which tyrosine-phosphorylated CagA affects signaling within gastric epithelial cells. Because the C-EPIYA ؉ (phosphorylated) CagA (from 147C) has greater binding affinity to SHP2, as shown previously (23,34,36) and confirmed in our co-immunoprecipitation experiments, the extent of this interaction could affect downstream signaling pathways. For example, SHP2 positively regulates ERK activity, which is necessary for CagA induction of the hummingbird phenotype (23).…”

Section: Discussionmentioning

confidence: 64%

“…Expression Vectors-SHP2-specific small interfering RNA (siRNA) (kindly provided by Prof. M. Hatakeyama) was used to silence the expression of SHP2 (34). To selectively knock down human SHP2 expression, AGS cells were co-transfected with pSUPER-SHP2 and pBabePuro.…”

Section: Methodsmentioning

confidence: 99%

Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells

Lee

Kim

Choi

et al. 2010

Journal of Biological Chemistry

119

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The Helicobacter pylori protein CagA may undergo tyrosine phosphorylation following its entry into human gastric epithelial cells with downstream effects on signal transduction. Disruption of the gp130 receptor that modulates the balance of the SHP2/ERK and JAK/STAT pathways enhanced peptic ulceration and gastric cancer in gp130 knock-out mice. In this study, we evaluated the effect of translocated CagA in relation to its tyrosine phosphorylation status on the gp130-mediated signal switch between the SHP2/ERK and JAK/STAT3 pathways. We showed that in the presence of CagA, SHP2 was recruited to gp130. Phosphorylated CagA showed enhanced SHP2 binding activity and ERK1/2 phosphorylation, whereas unphosphorylated CagA showed preferential STAT3 activation. These findings indicate that the phosphorylation status of CagA affects the signal switch between the SHP2/ERK and JAK/STAT3 pathways through gp130, providing a novel mechanism to explain H. pylori signaling.Helicobacter pylori frequently colonizes the human stomach (1), and hosts infected by cagA-positive strains are at increased risk of gastric cancer and peptic ulceration (2-10). H. pylori injects the CagA protein into host gastric epithelial cells via a type IV secretion system (11-16). The injected CagA is tyrosine-phosphorylated by Src family protein-tyrosine kinases and binds SHP2 (Src homology 2 domain-containing Src homology tyrosine phosphatase) (17-21); the CagA-SHP2 complex has been detected in human gastric mucosa (22,23).The IL6/gp130/STAT3 (interleukin-6/glycoprotein 130/signal transducer and activation of transcription 3) pathway has been shown to play a role in the development of gastric cancer (24, 25). IL6 exerts its biological activities through the receptor subunit gp130 (26). At least two functional modules of gp130 have been characterized; one encompasses four membranedistal Tyr(P) binding sites for the Src homology 2 domain of the latent transcription factors, STAT1 and STAT3. The other comprises the membrane-proximal Tyr(P) 757 residue responsible for the engagement of cytoplasmic SHP2 (26, 27).IL6 induces recruitment and homodimerization of gp130, potentially leading to balanced signaling through both the JAK/ STAT and SHP2/Ras/ERK signaling pathways (28, 29). However, disrupting this balance in the gp130 "knock-in" mouse induced premalignant lesions, including atrophy, intestinal metaplasia, dysplasia, and ultimately gastric cancer (24). Similarly, when the IL6 cytokine family signaling pathway is disrupted, increased STAT3 signaling may favor development of gastric adenomas, whereas increased SHP2/ERK 2 signaling may lead to mucosal inflammation (25,30).That cellular factors that are up-regulated in response to H. pylori could modulate SHP2/ERK or JAK/STAT signaling pathways suggests that H. pylori persistence and its consequent pathologies could be influenced by gp130-mediated signal transduction through these pathways (30). In this study, we examined the role of CagA tyrosine phosphorylation status in the activation of the SHP2/ERK and...

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“…Accordingly, we next examined the effect of CagA on FAK tyrosine phosphorylation in AGSderived G11 cells, which stably express SHP-2-specific siRNA and thus show a marked reduction in SHP-2 expression (22). Notably, the level of FAK tyrosine phosphorylation was significantly elevated in G11 cells compared with the level in parental AGS cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…AGS human gastric epithelial cell and AGSderived stable transfectant clone G11, in which SHP-2 was constitutively knocked down by SHP-2-specific small interfering RNA (siRNA) (22), were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Expression vectors were transiently transfected into AGS cells by using Lipofectamine 2000 reagent (Invitrogen) as per the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Focal Adhesion Kinase Is a Substrate and Downstream Effector of SHP-2 Complexed with Helicobacter pylori CagA

Tsutsumi

Takahashi

Azuma

et al. 2006

Molecular and Cellular Biology

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Conditional gene silencing utilizing the lac repressor reveals a role of SHP‐2 in cagA‐positive Helicobacter pylori pathogenicity

Abstract: RNA interference (RNAi) is a newly described biological phenomenon mediated by small interfering RNA (siRNA) that targets mRNA for degradation by cellular enzymes and has become a powerful method for studying gene functions in mammalian systems

Cited by 54 publications

References 30 publications

Human gastrin mRNA expression up-regulated by Helicobacter pylori CagA through MEK/ERK and JAK2-signaling pathways in gastric cancer cells

Human gastrin mRNA expression up-regulated by Helicobacter pylori CagA through MEK/ERK and JAK2-signaling pathways in gastric cancer cells

Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells

Focal Adhesion Kinase Is a Substrate and Downstream Effector of SHP-2 Complexed with Helicobacter pylori CagA

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