To expand our knowledge of Helicobacter pylori virulence mechanisms, we used iTRAQ (isobaric tagging reagents for relative and absolute quantification)-based proteomic analysis to investigate the effect of H. pylori on gastric AGS tissue culture cells. In particular, we were interested in finding out which effects of H. pylori were dependent on the cytotoxins CagA and VacA. Protein analysis was restricted to detergent-resistant membranes (DRMs), because both toxins were described previously to localize in lipid raft-like domains. Using H. pylori wild type and two isogenic mutants, DeltacagA and DeltavacA, we identified a total of 21 proteins that were either increased or decreased in the DRMs due to bacterial infection. The effect on three of these proteins, ezrin, syndecan-4 and Rab11-FIP1, were furthermore dependent on CagA. Because these proteins have been implicated in cell migration, adhesion and polarity, they might act as important mediators of CagA cytotoxicity.

CagA of Helicobacter pylori alters the expression and cellular distribution of host proteins involved in cell signaling.

Helicobacter pylori (Hp) specific antigens were found deposited in the glomeruli in some kidney diseases. However, the underlying molecular mechanisms remain to be elucidated. The aim of this study was to investigate the effect of cytotoxin associated gene A protein (CagA), a key virulence factor of Hp, on mouse podocytes. Cells were cultured and treated with recombinant CagA protein. The expression of the tight junction protein ZO-1 and p38 MAPK signaling pathway activation were measured with real-time RT-PCR and western blotting. The filtration barrier function of podocytes was evaluated with albumin influx assay. CagA decreased the expression and membrane distribution of ZO-1, impaired the filtration barrier function of podocytes, while activating p38 MAPK signaling pathway in these cells. Selective p38 MAPK inhibition partly prevented CagA-induced filtration barrier dysfunction of podocytes through ameliorating ZO-1 downregulation. Taken together, the results suggested that CagA, at least via p38 MAPK signaling pathway, may induce podocyte injury. Anti-Hp therapy may be beneficial for the treatment of kidney diseases related to Hp antigen deposition.

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Helicobacter pylori cytotoxin-associated gene A impairs the filtration barrier function of podocytes via p38 MAPK signaling pathway.

................................................................................................... i CONTENTS..................................................................................................iv ABBREVIATIONS ........................................................................................1 LITERATURE REVIEW...............................................................................3 Introduction.............................................................................................3 OPN in inflammatory response.............................................................7 OPN in cancer.........................................................................................9 Summary ...............................................................................................15 CHAPTER I. ................................................................................................16 Abstract.................................................................................................17 Introduction...........................................................................................19 Materials and Methods ........................................................................22................................................................................................17 Introduction...........................................................................................19 Materials and Methods ........................................................................22 Mice............................................................................................................................................22 Cell cuture and isolation of peritoneal macrophages............................................22 Helicobacter pylori infection and in vitro co-culture...........................................23 Necropsy and histopathological examination...........................................................25 Immunohistochemistry (IHC) and double immunofluorescence....................26 Lentiviral shRNA for stable OPN knockdown.........................................................27 In vitro migration assay.....................................................................................................28 Quantitative real-time RT-PCR (QRT-PCR).....................................................29 Western blotting (WB)......................................................................................................30

마우스 위암과 간암 모델에서 Osteopontin의 역할 : The roles of osteopontin on gastric and hepatic carcinogenesis in mouse model

Gastric carcinoma (GC) remains one of the most malignant tumors either in morbidity or mortality rates around the world (IARC, 2002). Development of GC is influenced by multiple factors including genetic, biological, social, and psychological ones, etc.., however, the function mechanisms of which are too sophisticated and still under explorations (Matysiak-Budnik & Megraud, 2006). Similar to other malignant tumors, GC leads to death mainly due to system or organ failure because of cancer advancement and metastasis. Chemotherapy-based regimens combined with radiological and immunomodulating therapies are fundamental but unsatisfactory because most GC patients are in advanced stage when diagnosed, although radical operation throws those at early stage a light of prolonged survival and even clinical cure (Quiros & Bui, 2009). As for prophylaxis, many reports have mentioned the protective role of eradication of Helicobacter pylori, which has been identified as a definite carcinogen for GC, but few evidences proved a successful H. pylori vaccine showing effects on GC prevention, just as Hepatitis B virus vaccine on prevention of primary hepatocellular carcinoma (IARC, 1994; Murakami et al., 2005; Cai et al., 2005; Di Bisceglie 2009). H. pylori, a stomach colonizing spiral gram-negative bacterium, interacts with the host in a multiplicity of ways during its adhesion, colonization, invasion, and induction of inflammatory and immune responses (Peek 2005). The great majority of researchers link H. pylori infection with development or even recurrence of GC according to some clinical trials, meta-analyses, and in vitro experiments (Wong et al., 2004; Fukase et al., 2008). However, a few recent studies have disclosed the other side of the coin, in which positive H. pylori status appears to be associated with better outlook in GC patients (Meimarakis et al., 2006; Marrelli et al., 2009). Therefore, H. pylori probably factually play a bi-directional role in GC just like a double-edged sword. To learn about both edges of H. pylori infection will provides us with new sights in vaccine design, prevention, and even therapy of GC. Herein, we try to re-elucidate the relationship between H. pylori and GC from novel angles, in which GC consists of H. pylori-related (Hp-GC) and non-H. pylori-related (nHp-GC) ones,

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A Double-Edged Sword: Roles of Helicobacter Pylori in Gastric Carcinoma

/pdf/the-roles-of-osteopontin-on-gastric-and-hepatic-2y331vv9p2.pdf

沙織津田

Papers

Helicobacter pylori CagA はTGFβ-smad 経路のシグナル伝達を抑制し, TGFβによる胃粘膜上皮細胞の増殖抑制を回復させる

沙織 津田

Papers

Helicobacter pylori CagA はTGFβ-smad 経路のシグナル伝達を抑制し, TGFβによる胃粘膜上皮細胞の増殖抑制を回復させる

沙織津田