Exploring the relationship between Helicobacter pylori and host cell invasion: from phenotypes to molecular mechanisms

Doutoramento em Biomedicina Faculdade de Medicina da Universidade do Porto Exploring the relationship between Helicobacter pylori and host cell invasion: from phenotypes to molecular mechanisms Relação entre Helicobacter pylori e invasão das células do hospedeiro: dos fenótipos aos mecanismos moleculares Ângela Margarida Amorim Costa Porto, Por t ugal 2012 Supervisor: Prof. Céu Figueiredo (University of Porto) Co-Supervisor: Prof. Thomas F Meyer (Max Planck Institute for Infection Biology) Thesis committee: Prof. Agostinho Marques (University of Porto) Prof. Bianca Bauer (Max Planck Institute for Infection Biology) Prof. Peter Jordan (National institute of Health” Dr. Ricardo Jorge”) Prof. Manuel Sobrinho Simões (University of Porto) Prof. Raquel Seruca (University of Porto) Prof. Mário Dinis Ferreira (University of Porto) Prof. Céu Figueiredo (University of Porto) On the front cover: Helicobacter pylori strain SS1. Adapted by permission from PLoS One (Marta Correia et al.) © 2012. Financial support by: SFRH/BD/36013/2007 i DISSERTAÇÃO DE CANDIDATURA AO GRAU DE DOUTOR EM BIOMEDICINA APRESENTADA À FACULDADE DE MEDICINA DA UNIVERSIDADE DO PORTO. ii Artigo 48º, § 3º - A Faculdade não responde pelas doutrinas expendidas na dissertação. (Regulamento da Faculdade de Medicina do Porto – Decreto-Lei Nº 19 337, de 29 de Janeiro de 1931). iii Este trabalho é dedicado à Célia e ao Ricardo, por me terem empurrado para a frente quando eu queria ter ficado pelo caminho “ Nunca me resignaria na profissão ao absurdo constante de semelhantes situações catastróficas, em que a teimosia de um micróbio ou a rebeldia de uma célula desafiavam caprichosamente todas as forças mobilizadas do engenho humano.” Miguel Torga in “A Criação do Mundo IV” iv AGRADECIMENTOS / ACKNOWLEDGEMETS v Um doutoramento é muito mais que a tese ou a defesa, é um conjunto imenso de alegrias e frustrações que escritas dariam um livro bem maior que este. Para que o meu percurso de estudante de doutoramento (e de pré-estudante de doutoramento como BIC nos Pyloris) chegasse a bom termo tive a ajuda de muita gente, a quem tenho a obrigação de agradecer! Desde logo gostava de agradecer a toda a comunidade do IPATIMUP, e ao seu Director, Professor Sobrinho, por ter tornado tudo isto possível. Durante estes anos o IPATIMUP foi a minha segunda casa, não só pela quantidade de tempo lá passada, mas sobretudo por sentir que faço parte de uma Família. Podemos não publicar na Sience e na Natura (ainda…), mas temos os melhores colegas e ambiente de trabalho que se pode encontrar. Desde logo um agradecimento especial ao Pessoal do Cancer Genetics por proporcionarem diariamente aquela dose de insanidade mental necessária para manter a sanidade! Queria deixar um agradecimento muito especial à minha orientadora Céu, por me ter dado a oportunidade de trabalhar no seu Grupo, e aí fazer o meu trabalho de doutoramento. Obrigada pelas discussões científicas, por todo o teu apoio, por aturares o meu mau feitio, e por compreenderes que por detrás de um cientista está sempre uma pessoa “normal”. Céu és uma pessoa que admiro muito. A todos os meus coleguinhas do grupo do Helicobacter, os de hoje e os de ontem, pela amizade e pelos conhecimentos científicos, uma muito obrigada! Um agradecimento à Ana Costa por me ter recebido no Grupo, ao Nuno Guimarães um muito obrigada especial por desencantares os artigos a que mais ninguém tem acesso, e também pelas descobertas não científicas, mas não menos espectaculares, de músicas que não lembram nem ao Menino Jesus. À Ana Machado que apareceu como colega e foi para as Dinamarcas como amiga, ao Rui pela paciência, apoio, disponibilidade com a bicheza e por me ter ensinado a fazer Real-times. À Marina pelas discussões científicas. E aos caçulas Miguel e Inês por me aturarem nos últimos tempos. E agora um agradecimento mesmo muito especial às ex-pyloris e agora inebianas Maria e Marta! À Maria porque para além de ser colega é também amiga, e é uma pessoa extraordinária com quem podemos aprender a ser melhores pessoas e cientistas, e porque é a minha mãe científica na Biologia Celular. Muito do que sou no laboratório devo-o a ti. Um obrigado também por conseguires ver coisas para além do óbvio e conseguires entusiasmar as pessoas com resultados que à primeira vista parecem uma caca. À Marta tenho a LIST OF ABBREVIATIONS xii GPCR G-protein-coupled receptors Grb2 growth factor receptor-bound protein 2 GSK3β glycogen synthase kinase 3β GTP guanosine-5’-triphosphate hBD3 human beta-defensin 3 HB-EGF heparin binding epidermal growth factor HER human epidermal growth factor receptor HGF hepatocyte growth factor H. pylori Helicobacter pylori HRP horseradish peroxidase HtrA high temperature requirement A IF immunofluorescence IFN-γ interferon γ IGFBP insulin-like growth factor binding protein IHC immunohistochemistry IL interleukin IL-1ra interleukin-1 receptor antagonist Ile isoleucine IP immunoprecipitation IQGAP IQ motif containing GTPase activating protein JAM junctional adhesion molecule JNK c-Jun N-terminal kinase Kb kilobase kDa kiloDalton LIME Lck-interacting membrane kinase LRP6 low density lipoprotein receptor-related protein 6 MALT mucosal-associated lymphoid tissue MLC myosin light chain MMP metalloproteinase MOI multiplicity of infection mRNA messenger RNA MUC mucin MW molecular weight N-cadherin neuronal cadherin LIST OF ABBREVIATIONS xiii NF-kB nuclear factor kappa-light-chain-enhancer of activated B cells Nod1 nucleotide-binding oligomerization domain protein 1 NTAL non-T cell activation linker N-terminal amino terminal PDFG platelet derived growth factor PDFGR platelet derived growth factor receptor PI3K phosphatidylinositol 3-kinase PLC-γ phospholipase C γ Pro proline PTP1B protein tyrosine phosphatase 1B OipA outer inflammatory protein A PBS phosphate buffered saline PKC protein kinase C PP1α phosphoprotein phosphatase 1α P-Tyr phospho tyrosine RNA ribonucleic acid RPMI Roswell Park Memorial Institute medium RTK receptor tyrosine kinase qRT-PCR quantitative real time polymerase chain reaction SabA sialyl-Lewis X binding protein A SD standard deviation SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SE standard error SF scatter factor SH2 Src homology 2 siRNA small interference RNA STAT3 signal transducer and activator of transcription 3 T4SS type IV secretion system TCF-LEF T-cell factor-lymphoid enhancer factor TGF transforming growth factor TIMP tissue inhibitor of metalloproteinase Tir Translocated intimin receptor TNF-α tumor necrosis factor α TSA trypticase soy agar LIST OF ABBREVIATIONS xiv Tyr tyrosine vacA vacuolating cytotoxin gene A WB western blot ZO-1 zonula occludens xvii LIST OF PUBLICATIONS ………………………………………………………………………………... iii LIST OF ABBREVIATIONS ……………………………………………………………………............... v TABLE OF CONTENTS ………………………………………………………………………………….. xi INTRODUCTION …………………………………………………………………………………………... 1 1. Helicobacter pylori ……………………………………………………………………………………... 3 1.1. H. pylori virulence factors ………………………………………………………………………… 4 1.2. Clinical outcomes of H. pylori infection ………………………………………………………….. 6 1.2.1. H. pylori-associated gastric carcinoma ………………………………………………….. 6 1.3. H. pylori and host epithelial cell-cell junctions ……………………………………………………… 7 1.3.1. H. pylori and adherens junctions disruption ……………………………………………...... 8 H. pylori and E-cadherin ……………............................................................................................ 10 H. pylori and β-catenin ................................................................................................................. 12 H. pylori and p120-catenin .............................................................................................................. 13 2. H. pylori infection and epithelial cell signaling …………………………………………………... 14 2.1. c-Met signaling pathway ………………………………………………………............................ 15 2.2. EGFR signaling pathway …………………………………………………………………………... 17 3. Metalloproteinases …………………………………………………………………......................... 19 3.1. H. pylori infection and MMPs ……………………………………………………………............... 24 OUTLINE AND AIMS ……………………………………………………………………………………... 27 MATERIALS AND METHODS …………………………………………………………………………... 31 1. Cell culture ………………………………………………………………………………………...... 33 2. H. pylori strains and growth conditions ………………………………...................................... 33 3. Infection of gastric cells ……………………………………………………………………………. 34 4. Invasion assay …………………………………………………………………………................. 34 5. Small interference RNA (siRNA) transfection ………………………………………………….. 35 6. Cell treatment with growth factors, cytokines and chemical inhibitors ………………………… 35 7. Preparation of cell lysates and co-immunoprecipitation ………………………………………. 36 8. Immunoblot analysis ……………………………………………………………………………….. 36 9. Antibodies ………………………………………………………………………………………....... 37 10. Quantification of mRNA by real-time PCR ……………………………………………………….. 38 11. Preparation of conditioned medium …………………………………………………………… 39 12. MMP-10 activity assay ……………………………………………………………………………. 39 13. Immunocitochemistry and confocal microscopy ……………………………………………….. 39 14. Transduction of AGS cell line with human E-cadherin cDNA ………………………………… 40 15. Aggregation assay …………………………………………………………………………………. 40 16. Statistical analysis …………………………………………………………………………………. 41 RESULTS …………………………………………………………………………………………………. 43 Part I 45 1. Analysis of the effects of H. pylori in host gastric epithelial cell invasion ………………………..... 47 1.1. Role of H. pylori and bacterial virulence factors in host cell invasion ………………………. 47 1.2. Role of c-Met receptor in H. pylori-mediated cell invasion …………………………………...... 48 1.3. Role of c-Met downstream targets in H. pylori-mediated cell invasion ………………………… 49 1.4. Role of H. pylori and of c-Met in Nck, c-Src, and PLC-γ activation …………………………… 52 2. Analysis of the effect of H. pylori in modulation of matrix metalloproteinase 10 (MMP-10)……..... 55 2.1. Up-regulation of MMPs by H. pylori………………………………………………………………… 55 2.2. Analysis of the effect of H. pylori in MMP-10 protein secretion and activity ……………… 56 2.3. Role of bacterial virulence factors in H. pylori-induced MMP-10 expression ………………. 57 2.4. MMP-10 is involved in H. pylori-mediated cell invasion ……………………………………… 59 2.5. Role of c-Met and EGFR in H. pylori-induced MMP-10 expression………………………... 60 2.6. Role of c-Met and EGFR downstream targets in H. pylori-induced MMP-10 expression.. 62 2.7. Analysis of the participation of ERK1/2, JNK, and p38 signaling pathways in H. pyloriinduced MMP-10 expression …………………………………………………………………….. 63 2.8. Analysis of MMP-10 modulation by cytokines produced by H. pylori infection…………… 64 2.9. Role of MMP-10 in MMP-1 activation in H. pylori infection ………………………………….. 65 Part II 67 xix 1. Analysis of the effects of E-cadherin in H. pylori-mediated cell invasion and signaling …... 69 1.1. H. pylori-mediated invasive phenotype in cells with intact adherens junctions ………….. 69 1.2. Generation and characterization of the AGSEcad cell line ………………………………..... 70 1.3. Role of E-cadherin in H. pylori-mediated cell invasion ………………………………………. 72 1.4. Role of E-cadherin in H. pylori-mediated c-Met and p120-catenin tyrosine phosphorylation…………………………………………………………………………………..... 72 1.5. Effect of H. pylori on the localization of elements of the E-cadherin-catenin complex ….. 74 1.6. Analysis of H. pylori CagA interactions with E-cadherin and with p120-catenin ………………. 75 DISCUSSION ………………………………………………………………………………………………. 81 Part I. Analysis of the effects of H. pylori in host gastric epithelial cell invasion and in modulation of matrix metalloproteinase 10 (MMP-10)……... …………………………………………………………… 83 Part II. Analysis of the effects of E-cadherin in H. pylori-mediated cell invasion and signaling ……… 90 Concluding remarks ……………………………………………………………………………................. 94 REFERENCES ……………………………………………………………………………………………. 97 SUMMARY ………………………………………………………………………………………………… 117 SUMÁRIO ………………………………………………………………………………………………..... 121 PAPERS ………………………………………………………………………………………………….. .. 125 INTRODUCTION INTRODUCTION 8 molecular mechanisms behind this process are not well known. H. pylori affects the epithelium in a multistep degenerative process that starts with the inflammation associated with the gastric colonization, with relevance for the action of the interleukins and reactive oxygen and nitrogen species produced in that context. At the same time, H. pylori promotes epithelial damage at the site of attachment on the host cell, both by altering the cell to cell adhesion and by the production of urease, lipases and toxins. In these conditions, the subepithelial tissue and the extracellular matrix are exposed, promoting the formation of ulcers. The disruption of epithelial junctions by H. pylori leads to a cell scattering phenotype, and causes cell elongation and increased motility (Montecucco et al., 2001). These alterations in the epithelium can lead to the disruption of the balance between epithelial cell proliferation and apoptosis (Cover et al., 2003). Indeed H. pylori has been associated with increased host cell apoptosis and with increased proliferation (Moss et al., 1996, Brenes et al., 1993). Additionally, H. pylori infection has been associated with DNA damage. This may occur either by the direct action of H. pylori virulence factors or by indirect action of reactive oxygen and nitrogen species produced by inflammatory cells (Smoot et al., 2000, Kim et al., 2002b, Maeda et al., 2002). In agreement with these observations, it has been shown that H. pylori downregulates mismatch repair systems of the host cells (Kim et al., 2002a, Machado et al., 2009). Overall, alterations in the balance of epithelial cell apoptosis and compensatory hyperproliferation induced by H. pylori may render these cells more susceptible to acquire mutations which may not be repaired, and therefore have increased risk for malignant transformation. Figure 3. Host, environmental and bacterial factors involved in the cascade leading to gastric carcinoma. Adapted by permission from Gastroenterology (Ernst et al.) © 2006. INTRODUCTION 9 1.3. H. pylori and host epithelial cell-cell junctions The gastric epithelium functions as a physical barrier, and seal the lumen preventing gastric acid and pathogens to reach the interstitial space and the blood stream. Epithelial cells are polarized structures linked to each other and to the extracellular matrix by cellular junctions. The function of the epithelium is mainly accomplished by the existence of these cell-cell junctions – tight junctions, adherens junctions, desmosomes, and gap junctions – which are essential components of epithelial integrity (Moens et al., Turnberg, 1985) (Figure 4). Defects of the junctional complexes are characteristic of several diseases, including cancer. The loss of cell-cell adhesion may induce loss of contact inhibition (Pignatelli et al., 1994), contributing to the early phases of malignant transformation of epithelial cells. Figure 4. Organization of the intercellular junctions. Epithelia are composed of a layer of cells attached to the basement membranes by focal contacts and to the adjacent cells by tight junctions, adherens junctions, and desmosomes. Adapted by permission from Cell (Perez-Moreno et al.) © 2003. The tight junctions are the most apical set of cell-cell junctions that separate the apical and the basolateral domains of the plasma membrane. Tight junctions functions, as a barrier that impairs the mixing of lipids and proteins from the apical and basolateral parts of the epithelial cells, and at the same time, control the paracellular flux of ions and small molecules (Tsukita et al., 2001, Balda et al., 2008). They are composed of transmembrane proteins occludin, claudins, and junctional adhesion molecules (JAMs), and cytosolic proteins, zona ocludens (ZO)-1, -2, and 3, that bridge transmembrane INTRODUCTION 10 proteins with the cytoskeleton (Tsukita et al., 2001). It has been shown that H. pylori infection of epithelial cells induces internalization of occludin and claudins, and increases myosin light chain phosphorylation, leading to tight junction relaxation and increased epithelial barrier permeability (Wroblewski et al., 2009, Fedwick et al., 2005, Lapointe et al., 2010). Further, CagA interacts with ZO-1 and JAM-A, and recruits these proteins to the sites of bacteria attachment, altering the composition and function of the tightjunctional complex (Amieva et al., 2003). The VacA toxin was also described to decrease the transepithelial resistance, a process mediated by the tight junctions complexes (Papini et al., 1998, Pelicic et al., 1999). Gap junctions are formed by channels constituted by connexins that allow intercellular passage of ions and small molecules such as calcium, ATP and cAMP (Laird, 2006). In addition, gap junctions also have a role in regulating cell morphology, establishing polarity, and rearrangement of the cytoskeleton (Matsuuchi et al., 2012). There are few reports addressing the role of H. pylori in gap junction modulation. Both CagA-positive and negative strains affect the gap-junctional function, with a more significantly effect in the case of CagA-positive strains (Tao et al., 2007, Xu et al., 2008), and bacteria eradication leads to promotion of cell junction formation (Xu et al., 2008). Desmosomes provide mechanical stability and intercellular communication to neighboring cells. They are composed by transmembrane desmoglein and desmocollin cadherins, which bind cytoplasmatic plakoglobin and plakophilin, which in turn bind to desmoplakin. The latter anchors intermediate filaments, which establish a mechanical continuum across cells (Odland, 1958, Matter et al., 2003, Johnson, 2005). The infection with H. pylori resulted in no alteration in the components of the desmosomes, in patients with gastroesophageal reflux disease (Wex et al.). 1.3.1. H. pylori and adherens junctions disruption Adherens junctions are localized immediately below tight junctions and their main function is to maintain cell-cell adhesion. The major component of the adherens junctions is the transmembrane protein E-cadherin. The extracellular part of E-cadherin establishes homophilic interactions with E-cadherin molecules of neighboring cells, promoting cohesion of the epithelium. E-cadherin may also establish heterophilic interactions, namely with the receptor tyrosine kinases EGFR and c-Met (Hoschuetzky et al., 1994, Mateus et al., 2007, Hiscox et al., 1999, Reshetnikova et al., 2007), modulating their signaling properties. The cytoplasmic domain of E-cadherin is associated with β-, INTRODUCTION 11 p120-, and α-catenins, and the latter establishes a connection between E-cadherin and the actin cytoskeleton (Figure 5). These protein-protein interactions, as well as the phosphorylation status of the catenins, are important in junction stabilization (Reynolds et al., 2004b, Yanagisawa et al., 2006). The alterations in the phosphorylation status of the adherens junctions components can be mediated by RTKs such as EGFR, c-Met and FGFR or by the non-receptor tyrosine kinase c-Src, resulting in the disassembly of the cytoplasmatic adhesion complex, disruption of cadherin-mediated cell adhesion and cell scattering (Behrens et al., 1993, Hamaguchi et al., 1993, Fujita et al., 2002). Apart from the adhesive function, adherens junctions are important in regulation and maintenance of cellular polarity, actin cytoskeleton and organelles organization, motility, tight junction formation, signal transduction, inhibition of proliferation, prevention of apoptosis, and are important to suppress epithelial cell invasion and metastatic spread in a cancer context. In the absence of E-cadherin the other members of the complex are not able to promote cell-cell adhesion (Jamora et al., 2002, Bilder et al., 2000). Figure 5. Schematic representation of the adherens junction complex. Adapted by permission from Nature Reviews Genetics (Fuchs and Raghavan) © 2002. Disruption of the intercellular junctions is a strategy used by several microorganisms as a means of adhering to, entering cells, and/or exploiting host signaling to their advantage. Although the first line of defense against infectious agents in mucosal epithelia are tight junctions, microorganisms also explore cell-cell junctions at levels below the tight junctions. Indeed, E-cadherin is used as a receptor for adhesion and/or internalization by INTRODUCTION 12 several microorganisms, allowing microbial persistence in the host, avoidance of mechanical clearance, and increased pathogenesis (Mengaud et al., 1996, Lecuit et al., 2000, Lecuit et al., 2001, Phan et al., 2007, Wu et al., 1998, Katz et al., 2000, Inoshima et al., 2011). Several lines of evidence show that H. pylori interferes with cell-cell junctional complexes, although the importance of this phenomenon for gastric disease development is not fully understood. The known mechanisms underlying H. pylori-mediated adherens junctions disruption are diverse and directed towards different components of the complex. H. pylori and E-cadherin Studies have shown that H. pylori targets E-cadherin by several mechanisms. Promoter methylation of the CDH1 gene (encoding E-cadherin) is frequently observed in H. pyloriinfected individuals (Chan et al., 2003a, Leung et al., 2006, Perri et al., 2007). The importance of this epigenetic mechanism of gene silencing in the context of H. pylori infection is reinforced by studies showing that eradication of H. pylori reduces CDH1 promoter methylation levels (Leung et al., 2006, Perri et al., 2007, Chan et al., 2006). Although the molecular mechanisms through which H. pylori mediates E-cadherin promoter methylation are not fully understood, Qian et al. have shown that CDH1 promoter methylation could be induced by infection with H. pylori or treatment of gastric cancer cell lines with interleukin-1β (IL-1β), a cytokine up-regulated in the context of H. pylori infection (Qian et al., 2008). This effect was inhibited by treatment with an interleukin-1 receptor antagonist (IL-1ra) antibody, suggesting that IL-1β may play a role in E-cadherin methylation (Qian et al., 2008). More recently, the same group has extended the study and showed that H. pyloriand IL-1β-mediated CDH1 promoter methylation, led to decreased E-cadherin expression and concomitant increase in DNA methyltransferase activity (Huang et al., 2012). Nevertheless, other studies have failed to show a decrease in E-cadherin expression associated with H. pylori infection both in cell lines and in the gastric mucosa (Bebb et al., 2006, Conlin et al., 2004, Chan et al., 2003a). Of note, no CDH1 mutations have been described associated to H. pylori infection. In addition to epigenetic silencing, H. pylori has been associated with other mechanisms of disturbance of E-cadherin functions, such as proteolytic cleavage of its extracellular domain (also known as ectodomain shedding), and protein delocalization from the cell membrane. INTRODUCTION 13 Weidig et al. and Schirmeister et al. reported E-cadherin ectodomain shedding upon H. pylori infection in two different cell line models, the breast cancer MCF-7 and the gastric cancer NCI-N87 cells, respectively (Schirrmeister et al., 2009, Weydig et al., 2007). In both reports, ectodomain shedding of E-cadherin was neither dependent on H. pylori CagA nor on the presence of a functional T4SS. This finding is in keeping with a recent report showing that H. pylori-positive patients had significantly higher serum levels of soluble E-cadherin than uninfected controls, independently of the CagA status of the infecting strain (O'Connor et al.). There is evidence that the host disintegrin metalloproteinase ADAM10 contributes to H. pylori-induced shedding of E-cadherin in NCI-N87 cells, but since specific inhibition of ADAM10 led to a partial inhibition of Ecadherin shedding, it is possible that other proteases are also involved in this process (Schirrmeister et al., 2009). In fact, Hoy et al. have recently identified the hightemperature requirement A (HtrA), a serine protease from H. pylori, as a new secreted virulence factor which cleaves the ectodomain of the E-cadherin. E-cadherin shedding by HtrA leads to epithelial barrier disruption and may allow H. pylori to access the intercellular spaces (Hoy et al., 2010). HtrA-mediated E-cadherin cleavage may be a pathogenic mechanism of multiple Gram-negative bacteria, as is has recently been shown for pathogens such as enteropathogenic Escherichia coli, Shigella flexneri, and Campylobacter jejuni (Hoy et al., 2012). Studies have shown that H. pylori also induces E-cadherin translocation from the cell membrane to the cytoplasm (Schirrmeister et al., 2009, Conlin et al., 2004). Conlin et al. have demonstrated that H. pylori-induced redistribution of E-cadherin to intracellular vesicles was accompanied by the translocation from the cytoplasm to intracellular tubular structures of IQGAP-1, a protein that regulates the formation of adherens junctions. The results of E-cadherin internalization mediated by H. pylori in the NCI-N87 gastric cell line were confirmed in primary gastric cells. After 48 hours of bacterial infection, the presence of a reduced level of E-cadherin at the cell membrane was demonstrated, mediating a reduced level of cell-cell adhesion. These alterations to epithelial cell adhesion molecules induced by H. pylori were paralleled by increased levels of Rho-GTP and cell migration (Conlin et al., 2004). Using the human breast cancer MCF-7 cell line, Weidig et al. also reported internalization of E-cadherin upon H. pylori infection, simultaneously with Ecadherin cleavage (Weydig et al., 2007). A rapid dissociation of the E-cadherin/βcatenin/p120 complex from the actin cytoskeleton was observed upon infection, by disruption of the interaction between E-cadherin and α-catenin (Weydig et al., 2007). Although E-cadherin alterations mediated by H. pylori are independent of CagA, physical interaction between these proteins has been described (Murata-Kamiya et al., 2007). INTRODUCTION 14 Murata-Kamiya et al. used the gastric cancer cell line MKN28 transfected with CagA expression vectors, and showed that CagA was able to interact with E-cadherin. This led to β-catenin release from the adherens junctions and nuclear translocation, with transactivation of genes encoding intestinal specific proteins like MUC2, contributing to the development of intestinal metaplasia (Murata-Kamiya et al., 2007). This effect was independent of the CagA tyrosine phosphorylation status but dependent on a specific region of CagA known as the multimerization sequence (Kurashima et al., 2008). H. pylori and β-catenin The binding of catenins to the intracellular domain of E-cadherin has a crucial role in the adherens junctional complex stabilization, connection to the cell cytoskeleton, and cell signaling. When not tethered at the membrane in the adherens junctions, β-catenin is phosphorylated by a multiproteic complex comprising the serine/threonine kinase glycogen synthase kinase 3β (GSK3β), and the scaffolding proteins adenomatous polyposis coli (APC), axin, and casein kinase 1α (Ck1α) and targeted to degradation via the ubiquitin/proteasome pathway (Aberle et al., 1997, Gooding et al., 2004). This degradation pathway is counteracted when the Wnt pathway is active, resulting in βcatenin stabilization in the cytoplasm. Cytoplasmic stabilized β-catenin may be translocated into the nucleus where it forms a nuclear complex with the transcription factors of the T cell factor⁄lymphoid enhancer-binding factor (TCF/LEF) family, promoting the expression of a wide-range of genes important for carcinogenesis (Gooding et al., 2004, Bierie et al., 2003, Marchenko et al., 2002, Polakis, 2000). Several studies have addressed the effect of H. pylori infection in β-catenin signaling and have shown that H. pylori leads to the delocalization of β-catenin from the cell membrane (Bebb et al., 2006, Weydig et al., 2007, Suzuki et al., 2005, Franco et al., 2005, MurataKamiya et al., 2007, Franco et al., 2008, Sokolova et al., 2008, Suzuki et al., 2009, Nakayama et al., 2009, Hung et al., 2009, Gnad et al., 2010, Kurashima et al., 2008). H. pylori phosphorylates and inactivates GSK3β (Tabassam et al., 2009, Sokolova et al., 2008, Nakayama et al., 2009, Suzuki et al., 2009) and, by suppressing GSK3β activity H. pylori leads to inhibition of β-catenin phosphorylation and ubiquitin-dependent degradation (Sokolova et al., 2008). H. pylori-mediated GSK3β suppression occurs through activation of the phosphatidylinositol 3-kinase⁄protein kinase B (PI3K) ⁄ (Akt) signaling pathway via EGFR and c-Met receptor (Nagy et al., 2011, Tabassam et al., 2009, Sokolova et al., 2008, Yan et al., 2009, Nakayama et al., 2009, Suzuki et al., 2009). INTRODUCTION 15 In one report however, β-catenin stabilization was described to occur in a phosphorylation-independent process (Franco et al., 2005). The canonical Wnt-signaling pathway appears to be involved in the regulation of βcatenin in the context of H. pylori infection. In fact, it has been shown that after H. pylori infection there is increased synthesis of Wnt10A by the epithelial cells (Kirikoshi et al., 2001). Additionally, H. pylori-induced activation of β-catenin was shown to involve the phosphorylation of the Wnt pathway co-receptor low density lipoprotein receptor-related protein LRP6 and proteins of the dishevelled family, namely Dvl2 and Dvl3 (Gnad et al., 2010). Several studies report β-catenin translocation to the nucleus after H. pylori infection (Suzuki et al., 2005, Franco et al., 2005, Murata-Kamiya et al., 2007, Franco et al., 2008, Sokolova et al., 2008, Suzuki et al., 2009, Nakayama et al., 2009, Hung et al., 2009, Gnad et al., 2010). There are however two studies in non-gastric cell lines that did not show nuclear translocation or activation of β-catenin signaling after infection (Bebb et al., 2006, Ogden et al., 2008). β-catenin nuclear translocation induced by H. pylori leads to TCF/LEF-dependent transcription of genes involved in carcinogenesis, including cyclin D1 (Murata-Kamiya et al., 2007, Sokolova et al., 2008, Nakayama et al., 2009, Gnad et al., 2010, Kurashima et al., 2008). This effect was shown to be CagA-dependent in the majority of studies (Suzuki et al., 2005, Franco et al., 2005, Murata-Kamiya et al., 2007, Suzuki et al., 2009, Hung et al., 2009, Kurashima et al., 2008), with the exception of one study performed in a non-human, non-gastric cell line (Sokolova et al., 2008). Additional effectors of the T4SS, like peptidoglycan (Nagy et al., 2011, Viala et al., 2004), and other H. pylori virulence factors, such as the multifunctional toxin VacA (Nakayama et al., 2009, Cover et al., 2005) and the outer membrane protein OipA (Franco et al., 2008, Yamaoka, 2011), may also be involved. The use of the Mongolian gerbil model has shown that H. pylori infection induces gastric carcinoma precursor lesions, with aberrant β-catenin expression, that led to transcriptional up-regulation of genes implicated in carcinogenesis. In this model-system, and in agreement with most of the cell-line models, aberrant βcatenin expression was associated with infection with H. pylori CagA-positive strains (Franco et al., 2005, Franco et al., 2008). H. pylori and p120-catenin p120-catenin (p120) is another important component in adherens junctional complex stability and signaling (Hartsock et al., 2008). p120 binds to the juxtamembrane domain of E-cadherin (Yap et al., 1998), and mutations in this domain abolish E-cadherin-mediated INTRODUCTION 16 cell-cell adhesion (Kaurah et al., 2007). In addition to E-cadherin stabilization, p120 can also interact with the transcription factor Kaiso (Daniel et al., 1999, van Hengel et al., 1999, Roczniak-Ferguson et al., 2003). Two reports show that H. pylori alters the cellular localization and the phosphorylation status of p120, but not total p120 protein levels (Krueger et al., 2007, Ogden et al., 2008). Krueger et al. showed that after six hours of infection of primary gastric epithelial cells, there was a recruitment of the non-phosphorylated p120 to perinuclear vesicles, whereas the fraction of phosphorylated p120 increased and could be detected in the nucleus, at the cell membrane, and at the leading edge of migrating cells (Krueger et al., 2007). Those alterations were associated with elongation of cells and increased migration. Ogden et al. also showed nuclear translocation of p120 after H. pylori infection of MKN28 cells and of ex vivo mouse gastric glands. Nuclear translocation of p120 induced by H. pylori was associated with increased MMP-7 mRNA, and occurred by release of the MMP-7 transcriptional repressor Kaiso. These changes were associated with reduced levels of p120 tyrosine phosphorylation, while total p120 remained unchanged (Ogden et al., 2008). H. pylori-mediated p120 nuclear translocation was dependent on factors of the T4SS, but independent of the T4SS effector CagA (Ogden et al., 2008). 2. H. pylori infection and host epithelial cell signaling Due to its close interaction with host cells, H. pylori has the capacity to induce alterations in the normal host cell signaling pathways. The alterations in the normal cell signaling by H. pylori can result in the induction of apoptosis, morphological changes, like the establishment of the “hummingbird phenotype,” disruption of the epithelial barrier, alteration in cell proliferation, and production of pro-inflammatory cytokines (Chen et al., 1997, Segal et al., 1999, Amieva et al., 2003, Fan et al., 1996, Crabtree et al., 1995) As a result of its localization at the cell membrane receptors tyrosine kinase (RTKs) are among of the first host cell molecules to be in contact with H. pylori during infection. The RTKs are transmembrane proteins that, typically, after activation by their ligands, dimerize or oligomerize (Lemmon et al., 1994, Lemmon et al., 1998, Hubbard et al., 2000, Schlessinger, 2000), making possible an efficient cross-phosphorylation of their adjacent cytoplasmic tyrosine kinase domains. The phosphorylation of the tyrosine residues, a characteristic of the transmembrane signaling, creates docking sites for several molecules involved in a plethora of signal transduction pathways. INTRODUCTION 17 Overexpression and mutations of the RTKs signaling pathways elements are associated with carcinogenesis and are targets for anticancer therapy (Brunelleschi et al., 2002). RTKs such as EGFR and c-Met are basolateral localized in polarized cells (Crepaldi et al., 1994, Maratos-Flier et al., 1987). After the disruption of cellular junctions mediated by H. pylori, the polarity of host cell is altered and the RTKs may be now localized apically, in a cellular localization more accessible to the bacteria. Because of these facts, the study of the H. pylori-mediated alterations in host cell signaling is critical to understand the outcomes of the infection. 2.1. c-Met signaling pathway The c-Met receptor is initially synthesized as a 170-kDa single chain intracellular precursor that after processing yields a mature 190 kDa disulfide-linked heterodimer form, which is cell surface-associated. The heterodimer is constituted of a 50 kDa αchain, entirely extracellular, and a 140 kDa β-chain, that spans the membrane, and consists of an extracellular domain, a membrane spanning domain, and a cytoplasmic tyrosine kinase domain (Giordano et al., 1989) (Figure 6). In a non-pathological context, c-Met activation by its natural ligand, the hepatocyte growth factor (HGF) also known as scatter factor (SF), occurs in a wide range of tissues, and is required for embryonic development (Uehara et al., 1995, Borowiak et al., 2004), liver regeneration (Borowiak et al., 2004, Huh et al., 2004), and wound healing (Chmielowiec et al., 2007). These cellular processes involve cytoskeleton reorganization, cell motility, proliferation, morphogenesis, invasion, and angiogenesis (Birchmeier et al., 2003, Jiang et al., 2005, Peruzzi et al., 2006). In a pathological context, abnormal c-Met signaling contributes to tumorigenesis, particularly to the invasive and metastatic phenotype (Furge et al., 2000). Deregulation of c-Met signaling by mutations, overexpression, and autocrine and paracrine activation, is associated with the severity of human cancers, including gastric carcinoma (Kuniyasu et al., 1992, Kaji et al., 1996, Amemiya et al., 2000, Birchmeier et al., 2003). It is known that the disruption of the c-Met multifunctional docking site impairs oncogenic transformation and invasive growth of tumor cells (Bardelli et al., 1998). In addition, c-Met phosphorylation stimulates epithelial to mesenchymal transition (EMT), a key feature of metastization (Birchmeier et al., 2003, Benvenuti et al., 2007). INTRODUCTION 24 3.1. H. pylori infection and MMPs H. pylori infection up-regulates the expression and activity of several MMPs both in vitro and in vivo. Although the molecular mechanisms that lead to MMP modulation are not well established, the role of MMPs in H. pylori-mediated cell invasion, migration and processing of growth factors has been reported (Wallasch et al., 2002, Wroblewski et al., 2003, Wu et al., 2005, McCaig et al., 2006, Oliveira et al., 2006, Yin et al., 2010). The majority of the data associating MMPs up-regulation and H. pylori infection came from analysis of cDNA arrays that compared infected versus non-infected conditions. These studies showed that H. pylori is associated with upregulation of all MMP subfamilies, namely MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP12, MMP-13, MMP-14, MMP-15, and MMP-19 (Yokoyama et al., 2000, Guillemin et al., 2002, Gooz et al., 2003, Kitadai et al., 2003, Mori et al., 2003, Wroblewski et al., 2003, Krueger et al., 2006). In addition, H. pylori itself is able to secrete a MMP-3 like enzyme (Windle et al., 1997, Gooz et al., 2001). Modulation of MMPs by H. pylori is best documented for MMP-1, MMP-7 and the gelatinases MMP-2 and MMP-9. It is reported that H. pylori up-regulate MMP-1 both in fibroblasts (Yokoyama et al., 2000) and in epithelial cells from infected individuals with gastritis and gastric cancer (Krueger et al., 2006, Wu et al., 2006, Pillinger et al., 2007). MMP-1 expression is dependent on the T4SS, OipA (Krueger et al., 2006, Wu et al., 2006), or CagA (Pillinger et al., 2007), depending on the cell line and strains used in different studies. MMP-1 up-regulation occurs in an ERK and JNK-dependent manner (Krueger et al., 2006, Pillinger et al., 2007), through activation of the Ras/Raf/RhoA/MAPK pathway (Wu et al., 2006), and under the regulation of PKC (Sokolova et al., 2012). The increase in MMP-1 in the context of H. pylori infection has been associated with increased basement membrane degradation and cell invasion (Krueger et al., 2006, Sokolova et al., 2012). H. pylori-mediated MMP-7 expression has been reported and the bacterial virulence factors VacA, T4SS, CagA, and OipA may play a role in this process (Bebb et al., 2003, Crawford et al., 2003, Wroblewski et al., 2003, Koyama, 2004, Yanagisawa et al., 2005, Chung et al., 2010, Eftang et al., 2012, Wu et al., 2006, Ogden et al., 2008). In vitro, MMP-7 is expressed in epithelial cells at the advancing edge of migrating cells (Wroblewski et al., 2003) and in vivo at the epithelial cells surface, and in the proliferative zone of the gastric glands (Wroblewski et al., 2003, Koyama, 2004, Bebb et al., 2003), and is also expressed in lymphocytes (Koyama, 2004). The signaling pathways underlying the H. pylori-mediated in MMP-7 expression are also controversial. H. pylori-associated cytokines IL-8, TNF-α and IL-6, as well as EGFR INTRODUCTION 25 ligands, may have a role in the process due to their ability to induce MMP-7 expression, and a role for gastrin has also been suggested (Wroblewski et al., 2003). MMP-7 expression occurs in a pathway involving Rho, Rac and the NF-kB (Wroblewski et al., 2003), in a ERK-dependent manner (Crawford et al., 2003), or through aberrant p120 catenin activation, that after nuclear translocation relieves the MMP-7 repressor Kaiso (Ogden et al., 2008). MMP-7 is involved in H. pylori-induced migration and invasion (Wroblewski et al., 2003), in the processing of insulin-like growth factor binding protein (IGFBP)-5 (McCaig et al., 2006) and in HB-EGF shedding (Yin et al., 2010). IGFBP-5 and HB-EGF have a role, respectively, in proliferation and migration of gastric myofibroblast (McCaig et al., 2006), and in EGFR activation and IL-8 production (Yin et al., 2010, Wallasch et al., 2002). H. pylori infection is associated with increased expression of the MMP-2 and MMP-9 gelatinases (Bergin et al., 2004, Kundu et al., 2006). MMP-2 and MMP-9 are expressed in epithelial cells and lymphocytes and MMP-9 is also expressed in fibroblast and macrophages (Koyama, 2004, Mori et al., 2003, Danese et al., 2004, Bergin et al., 2004). MMP-2 expression is induced by H. pylori in a CagA and T4SS-dependent manner (Yanagisawa et al., 2005), and is stimulated by the H. pylori-induced IL-21, in a process NF-kB dependent (Caruso et al., 2007). H. pylori-mediated MMP-9 expression is dependent on different bacterial virulence factors such as T4SS, CagA, OipA, HP-NAP, HpaA, urease, or LPS, depending on the cell line models and bacterial strains used (Yanagisawa et al., 2005, Mori et al., 2003, Wu et al., 2006, Nam et al., 2011, Kundu et al., 2006, Bergin et al., 2005). H. pylori-induced MMP-9 up-regulation in epithelial cells occurs via NF-kB (Mori et al., 2003, Nam et al., 2011, Wu et al., 2005, Caruso et al., 2007), COX-2 (Wu et al., 2005), CagA phosphorylation, SHP-2 and ERK (Nam et al., 2011). In addition, the infectionassociated cytokines TNF-α, IL-1β, IL-6, and IL-21 are able to induce MMP-9 secretion (Caruso et al., 2007, Kundu et al., 2006). Furthermore, H. pylori eradication leads to decreased MMP-9 in patients with chronic gastritis and gastric ulcers (Danese et al., 2004, Kubben et al., 2007, Kundu et al., 2011). H. pylori-induced increase in MMP-2 and MMP-9 activities results in increased cell invasion (Oliveira et al., 2006, Wu et al., 2005). 4 OUTLINE AND AIMS 6 OUTLINE AND AIMS 7 The general aim of the work presented in this thesis was to evaluate the role of H. pylori infection in the induction of host gastric epithelial cell invasion. The initial approach consisted on the evaluation of the invasive capacity through extracellular matrix components of the non-invasive human gastric cancer cell line AGS after infection with H. pylori. The role of the bacterial virulence factors CagA, T4SS, and VacA was also evaluated. After establishing that the c-Met receptor was important for H. pylori-mediated AGS cell invasion, the role of the downstream targets of c-Met was also elucidated. Additionally, and after demonstrating that Nck, PLC-γ, and c-Src were involved in H. pylori-mediated cell invasion, the activation of these molecules by H. pylori was studied. Studies in this section included in vitro infections of cell lines with H. pylori strains, followed by assessment of cell invasion in Matrigel assays. Studies also included transient transfection of cell lines with siRNAs for gene silencing, as well as co-immunoprecipitation and western blot analyses. Data obtained are presented in Part I.1 of the Results. Based on the results from Part I.1 pointing to a role of H. pylori in inducing extracellular matrix degradation by host cells, and since matrix metalloproteinases are important for that process, the next aim was to evaluate the role of H. pylori in MMP expression. Studies started with the validation of a panel of MMPs shown to be up-regulated in a cDNA expression microarray performed by our Group. After confirmation that H. pylori increased the expression of MMP-1, MMP-7, and MMP-10, a more detailed investigation of the effect of H. pylori on MMP-10 modulation was performed. Quantitative real-time polymerase chain reaction, western blot, and enzymatic activity assays were used in uninfected and H. pylori-infected cells. The role of the H. pylori virulence factors CagA and T4SS was addressed using bacterial mutants, and was further confirmed using a panel of H. pylori clinical isolates with known cagA and T4SS status. The involvement of specific host cell signaling pathways and of receptor tyrosine kinases in the modulation of MMP-10 expression by H. pylori was evaluated in experiments with siRNA gene silencing and with chemical inhibitors. The role of MMP-10 in H. pylori-mediated cell invasion was assessed with Matrigel assays.These results are presented in Part I.2 of the Results and in Paper I. Since the E-cadherin-catenin complex functions as an invasion suppressor, the next aim was to evaluate whether the complex had a role in H. pylori-mediated cell OUTLINE AND AIMS 8 invasion and signaling. For that, gastric cell lines with intact E-cadherin-catenin complex were used. Also, and for the purpose of comparison with previous data obtained in the AGS background, an AGS cell line with a wild-type E-cadherin was established. For the characterization of the structure and function of the adherens junctions, immunofluorescence, confocal microscopy and aggregation assays were used. The phosphorylation and interactions between proteins of the adherens junctions were evaluated by co-immunoprecipitation and western blot. The invasion capacity of the different cell lines was assessed with Matrigel assays. Data generated in this part of the thesis is presented in Part II of the Results and in Paper II. In this Thesis, a paper summarizing the findings of the literature regarding H. pylori infection and the adherens junctions (Paper III) and a paper reviewing the role of Eand P-cadherins in cancer (Paper IV) are also included, and have been partially used in the Introduction. MATERIALS AND METHODS MATERIALS AND METHODS MATERIALS AND METHODS 11 1. Cell cultures AGS (ATCC CRL-1739), NCI-N87 (ATCC CRL-5822), and IPA220 cells (Gartner et al., 1996), all derived from human gastric carcinomas were maintained in RPMI 1640 (Gibco), with 10% fetal bovine serum (FBS) (Hyclone), 2.5 µg/ml fungizone (Gibco), 200 µg/ml streptomycin, and 200 IU/ml penicillin (Gibco) at 37ºC, under a 5% CO2 humidified atmosphere. The AGSEcad cell line was maintained in the same conditions with supplementation with 5 µg/ml of blasticidin (Invitrogen) for clonal selection.The adherent cells were detached using trypsin (Gibco). 2. H. pylori strains and growth conditions Bacteria were grown in tryptic soy agar (TSA) supplemented with 5% sheep blood (BD Bioscience) and incubated 48 hours at 37ºC under a microaerophilic atmosphere. Experiments were performed with wild-type H.pylori strains 60190 (ATCC 49503), 26695 (ATCC 700392), 84183 (ATCC 53726), Tx30a (ATCC 51932), and G27 (Xiang et al., 1995), as well as with the mutant strains 60190∆vacA, 60190∆cagA, 60190∆cagE, 84183∆cagA and 84183∆cagE (all mutant strains were kindly provided by Professor John Atherton, Nottingham University, UK). A panel of clinical isolates with known vacA, cagA and cag PAI statuses was also used for some experiments (CI-7, CI-43, CI-45, CI-50, CI-51, CI-59, CI-60, CI-62, CI-63, and CI-69). Table I: Characterization of H. pylori strains in terms of virulence factors vacA, cagA and cag PAI Bacterial strain Virulence factor vacA cagA cag PAI 26695 s1/m1 + + 60190 s1/m 1 + + 60190∆cag A s1/m 1 - + 60190∆cag E s1/m 1 + - 60190∆vac A - + + 84183 s1/m1 + + MATERIALS AND METHODS 18 anti-rabbit-Alexa594 conjugated (Molecular Probes) were used as secondary antibodies, and incubated for 45 minutes. Coverslips were washed in PBS and mounted with Vectashield with DAPI (Vector Laboratories). A Leica DMRE2, a Zeiss Imager Z1 fluorescence microscope, or a SP2-SE-AOBS laser-scanning confocal microscope (Leica) was used. Confocal images were deconvolved with Huygens Pro3.2 (SVI, The Netherlands). 14. Transduction of the AGS cell line with human E-cadherin cDNA Plasmids containing human E-cadherin cDNA were constructed as previously described (Suriano et al., 2003b) and were stably transduced using a pLenti6/V5 expression vector (Invitrogen), according to manufacturer’s instructions. 293FT cell line, the lentivirus producer cell line, was maintained in DMEM medium (Gibco) at 37ºC, 5% CO 2 . After reaching the appropriate confluence the expression vector plus the ViraPower TM packaging mix were added in order to produce viral particles that were harvested after 24 hours. Posteriorly, these lentivirus were added to AGS cells and maintained for 24 hours. 5 µg/ml of blasticidin were added to the culture medium for clonal selection. Several clones were isolated, using cloning rings and splitting the cells surrounded by them, after which they were then transferred to new cell culture flasks. These clones were then characterized for the expression of Ecadherin by western blot and immunofluorescence. 15. Aggregation assay AGS and AGSEcad cells were trypsinized, resuspended as single-cells, and 2x105 cells in suspension were added to 96-well agar-coated plates (Bacto Agar). As control for Ecadherin-mediated aggregation, AGSEcad cells were incubated with an anti-E-cadherin antibody (MB2) (kindly provided by Professor Marc Bracke, Ghent University Hospital, Ghent, Belgium). Cells were then incubated for 48 hours at 37ºC in a 5% CO2 atmosphere. The ability of cells to aggregate or to persist as a single-cell suspension was evaluated microscopically and photographed. MATERIALS AND METHODS 19 16. Statistical analysis Data analysis were performed with Student’s t-test and expressed as mean values of at least three independent experiments ± standard errors, unless otherwise stated. The comparison between MMP-10 expression and the cagA status of the H. pylori clinical isolates was performed using the non-parametric Mann-Whitney test. For that, a variable that represents the average of the assays for each strain was created, and strains were divided into cagA-positive and cagA-negative groups. Differences were considered significant at P values less than 0.05. 20 R ESULTS 23 Part I RESULTS 25 Part I.1. Analysis of the effects of H. pylori in host gastric epithelial cell invasion The infection by H. pylori induces several well established alterations in the host cells, such as the induction of pro-inflammatory cytokines, enhancement of reactive oxygen and nitrogen species, and structural alterations in the organization of the cytoskeleton associated with increased motility and migration, that are named as “hummingbird phenotype” (Segal et al., 1999, Churin et al., 2001, Naumann et al., 2004). However, one of the less explored phenotypes that may be associated with the bacterial infection is host cell invasion. Invasive cells have the ability to degrade components of the extracellular matrix, usually by an up-regulation of MMPs, and to increase motility, that allows movement through the stromal tissue. The invasive phenotype must be regarded not only as general stromal tissue destruction, but also as a consequence of a complex signaling network that can also result in other cellular processes such as growth factor processing, cytokine release, and signaling transduction. 1.1. Role of H. pylori and bacterial virulence factors in host cell invasion As a first approach to evaluate the effects of H. pylori on epithelial cell invasion, AGS cells, derived from a gastric carcinoma and commonly used in H. pylori related studies, were used. Since the presence of virulence factors such as CagA, the cag PAI, and the VacA toxin are associated with most severe clinical outcomes related with H. pylori infection (Crabtree et al., 1991a, Blaser et al., 1995, Peek et al., 1995, Figueiredo et al., 2002), and since cell invasion may reflect cellular process that ultimately can lead to more severe disease, the role of these virulence factors in cell invasion was also evaluated. To try to understand this issue, a Matrigel invasion assay was used. In this assay, cells with invasive capacity are able to degrade an artificial matrix, Matrigel, which mimics the extracellular matrix. The cells that overcome that barrier can then be stained and counted. This assay was performed by incubating AGS cells alone, as a control, or with the H. pylori strains 26695, 60190, or G27, on Matrigel-coated filters for a period of 24 hours. In comparison with non-infected cells, H. pylori infected AGS cells showed a significant increase in the number of invasive cells through the Matrigel-coated filters (Figure 10A). RESULTS 26 To study the role of H. pylori virulence factors in the invasive phenotype, similar experiments were performed using the wild type strain 60190, and its mutants for cagA (60190∆cagA), cagE (60190∆cagE), and vacA (60190∆vacA). The co-culture of AGS cells with H. pylori wild type strain 60190 and the respective mutant strains that lack CagA or have impaired the T4SS (cagE mutant) showed that the number of invasive cells in the presence of these mutants was significantly lower than that observed in presence of the wild type strain (Figure 10B). Results also showed that the presence of the T4SS is more critical to the induction of the invasive phenotype than the presence of CagA, indicating that other virulence factors injected by the T4SS may also be involved in the process. The VacA virulence factor is not involved in the induction of the phenotype, since a strain that lacks this virulence factor induces similar levels of invasion compared to the wild type strain. AB Figure 10 – H. pylori stimulate AGS cell invasion in a T4SS dependent manner. Invasion assays on Matrigel coated filters for 24 h. (A) AGS cells infected with H. pylori strains 26695, 60190, and G27. (B) AGS infected with H. pylori wild type strain 60190, and its isogenic mutants 60190∆cagA, 60190∆cagE and 60190∆vacA. Data on graph represents the mean value +/- SE and are representative of three independent experiments. *, significantly different from non-infected cells;**, significantly different from cells infected with wild type H. pylori strain. 1.2. Role of the c-Met in H. pylori-mediated cell invasion Once established that H. pylori is able to induce an invasive phenotype in the host gastric cells, the focus of the research was directed to the molecular mechanisms behind this phenotype. In the cancer context, the RTK c-Met was shown to be a regulator of epithelial RESULTS 27 cell invasion (Hasegawa et al., 1995). Furthermore, this receptor was also shown to be activated by H. pylori infection leading to alterations in host cell motility (Churin et al., 2003). Therefore, the next experiments were performed to study the involvement of c-Met receptor in H. pylori-mediated invasive phenotype. For that, the chemical inhibitor NK4, an antagonist of the natural ligand of c-Met, the hepatocyte growth factor (HGF), as well as a siRNA targeting c-Met were used. AGS cells treated or not with NK4 or siRNA, and infected or not with H. pylori, were tested for the invasive capacities in Matrigel-coated filters. As depicted in the graphs of the Figure 11, both treatments with NK4 and with siRNA directed to c-Met resulted in a significant decrease in the number of invasive cells in the infected conditions. These results clearly point to a role of the c-Met receptor in the induction of invasion mediated by H. pylori. Figure 11 – c-Met has a role in H. pylori-mediated cell invasion. Non-treated AGS cells or treated with (A) NK4, a chemical inhibitors of c-Met, or (B) with a Non-silencing siRNA or an siRNA directed to c-Met, were co-cultured with H. pylori for 24 hours at a MOI of 100 on Matrigel coated filters. Data on graph represents the mean value +/- SE and are representative of three independent experiments. *, significantly different from non-infected cells;**, significantly different from cells infected with wild type H. pylori strain. 1.3. Role of the c-Met downstream targets in H. pylori-mediated cell invasion Results so far showed that H. pylori strains with a functional T4SS induce an increase in host cell invasion through the c-Met receptor. The next step was to identity the signaling molecules downstream c-Met that are implicated in H. pylori-mediated cell invasive phenotype. To address this issue a siRNA-based strategy was used. AGS cells were transiently transfected with siRNA abrogating the expression of the c-Met adaptors c-Cbl, Nck, Gab1, RESULTS 34 mRNA levels after infection with H. pylori, showing a 40-fold increase in expression in comparison with non-infected cells (Figure 14C). Figure 14. H. pylori increases in MMP mRNA expression of MMP-1, MMP-7, and MMP-10. AGS cells were infected with H. pylori strain 26695 for 24 hours at a MOI of 100. (A) MMP-1, (B) MMP-7, and (C) MMP-10 expression was analyzed by qRT-PCR. MMPs expression levels were normalized to GAPDH expression and results are presented as fold difference relative to uninfected cells. Data on graphs represents the mean value +/- SE and are representative of, at least, three independent experiments. *, significantly different from noninfected cells. 2.2. Analysis of the effect of H. pylori in MMP-10 protein secretion and activity Since MMP-10 showed the more pronounced increase in mRNA levels in the presence of H. pylori, and because the regulation of this metalloproteinase in the context of H. pylori infection had never been explored, attention was focused on this particular MMP. To assess whether the increase in MMP-10 mRNA after infection is accompanied by an increase in protein secretion, the conditioned medium of AGS cells infected for 24 hours was collected and concentrated, and the levels of secreted MMP-10 were analyzed by western blot. H. pylori significantly enhanced the secretion of MMP-10 into the medium. (Figure 15A). Of note, no MMP-10 secretion was observed in uninfected AGS cells. To address the enzymatic activity of secreted MMP-10, conditioned media of infected and non-infected AGS cells were analyzed using a fluorimetric assay. In this experiment, levels of active MMP-10 were calculated as a measure of cleavage of an enzyme substrate (FRET peptide with a specific sequence known to be cleaved by the active MMP-10), by changes in fluorescence emission. Compared with non-infected cells, cells RESULTS 35 infected with H. pylori showed a significant increase in emitted fluorescence, reflecting an increase in MMP-10 enzymatic activity (Figure 15B). Figure 15. H. pylori increases MMP-10 protein secretion and activity. AGS cells were infected with H. pylori strain 26695 for 24 hours at a MOI of 100. (A) Secreted MMP-10 was analyzed in conditioned media by western-blot and graphs represents the densitometry analysis of the blots. (B) MMP-10 activity in conditioned media was analyzed by FRET. Data on graphs represents the mean value +/- SE and are representative of three independent experiments. *, significantly different from non-infected cells. 2.3. Role of bacterial virulence in H. pylori-induced MMP-10 expression To define the contribution of the CagA and of the T4SS virulence factors in H. pylorimediated increase in MMP-10 mRNA, AGS cells were co-cultured for 24 hours with H. pylori strain 84183 and its cagA (84183∆cagA) and T4SS (84183∆cagE) mutants. The analysis of the mRNA levels of MMP-10 by qRTPCR showed that infection with wild type strain 84183 induces an increase in the expression of MMP-10 comparable to the one induced by H. pylori 26695 (Figure 16). In contrast, infection with the 84183∆cagA and 84183∆cagE mutants, lacking cagA and a functional T4SS, respectively, led to the expression of significantly less amounts of MMP-10 mRNA (Figure 16). These results show that the increase in MMP-10 expression induced by H. pylori is dependent on the presence of a functional T4SS. Since the T4SS is needed for CagA translocation into the host cell, and because the cagA mutant also induced low MMP-10 expression, it is likely that H. pylori-mediated up-regulation of MMP-10 is CagA-dependent (Figure 16). RESULTS 36 Figure 16. H. pylori-mediated MMP-10 expression is CagA dependent. AGS cells were infected with H. pylori for 24 hours at a MOI of 100 with wild-type strain 84183 and its cagE (84183∆cagE) and cagA (84183∆cagA) mutants. MMP-10 expression was analyzed by qRT-PCR. MMP-10 expression levels were normalized to GAPDH expression and results are presented as fold differences relative to uninfected cells. Data correspond to the mean values +/- SE and are representative of three independent experiments. *, significantly different from uninfected cells; **, significantly different from cells infected with wild-type H. pylori 84183. To confirm these results, AGS cells were infected for 24 hours with a panel of clinical isolates of H. pylori, with known cagA status, as well as with the non-pathogenic and cagA negative strain Tx30a. In general, H. pylori cagA-positive strains led to a more pronounced increase in MMP-10 expression after infection than did cagA-negative strains (Figure 17). The main exception was strain CI-50 that, although cagA-positive, was not able to significantly up-regulate MMP-10 (Figure 17). It is possible that this strain has an impaired T4SS and is not able to translocate CagA into the host cells. Curiously, Tx30a was the strain that among all cagA-negative strains induced a more elevated level of MMP-10, which was still, much less than those induced by cagA-positive strains (Figure 17). These results point to an important role of CagA in MMP-10 up-regulation mediated by H. pylor RESULTS 37 Figure 17. MMP-10 expression is higher in cells infected with H. pylori cagA-positive than with cagA-negative strains. AGS cells were infected for 24 hours at a MOI of 100 with a panel of H. pylori isolates with known cagA status. MMP-10 expression was analyzed by qRT-PCR. MMP-10 expression levels were normalized to GAPDH expression and results are presented as fold differences relative to uninfected cells. Data correspond to the mean values +/- SE and are representative of three independent experiments. *, significantly different from cagA negative strains. 2.4. MMP-10 is involved in H. pylori-mediated cell invasion As one of the main functions of the MMPs is the turnover and remodeling of the extracellular matrix (Parks et al., 2004), and since it was previously reported that H. pylori cag-positive strains induce gastric cell invasion in a context of increased MMP expression and activity (Oliveira et al., 2006, Sokolova et al., 2012), it was determined whether MMP10 is involved in cell invasion mediated by H. pylori. AGS cells were transiently transfected with a siRNA abrogating MMP-10 expression, and the efficiency of the knockdown was accessed by qRT-PCR. Invasion assays were performed 24 hours after transfection, by incubating AGS cells with H. pylori on Matrigel filters for an additional 24 hours. Silencing of MMP-10 inhibited H. pylori-induced MMP-10 expression in AGS cells (Figure 18A), and signifincantly inhibited cell invasion in response to H. pylori infection RESULTS 38 (Figure 18B). These findings demonstrate that MMP-10 plays a role in H. pylori-mediated cell invasion. Figure 18. MMP-10 is involved in H. pylori-mediated cell invasion. (A) AGS cells were treated with a Non-silencing siRNA or a siRNA directed to MMP-10, and co-cultured with H. pylori for 48 hours. The efficiency of the knockdown was evaluated by qRT-PCR. (B) AGS cells treated with a Non-silencing or a siRNA directed to MMP-10, were co-cultured with H. pylori for 24 hours at a MOI of 100 on Matrigel coated filters. Data on graph represents the mean value +/- SE and are representative of three independent experiments. *, significantly different from non-infected cells;**, significantly different from cells infected with H. pylori 60190. 2.5. Role of c-Met and EGFR in H. pylori-induced MMP-10 expression It has been reported that MMPs may be up-regulated by growth factors that activate RTKs (McCawley et al., 2001, Egeblad et al., 2002). c-Met and EGFR are two RTKs known to be activated during H. pylori infection (Wallasch et al., 2002, Churin et al., 2003, Oliveira et al., 2006). Additionally, EGFR was shown to be implicated in MMP-10 upregulation in squamous cell carcinoma of head and neck (Wilkins-Port et al., 2007). Therefore, the next experiments were performed to address whether c-Met and EGFR are implicated in H. pylori-mediated MMP-10 expression. First, it was tested whether stimulation of AGS cells with HGF and EGF, the natural ligands of c-Met and EGFR respectively, induced differences in MMP-10 expression. AGS cells were treated with HGF at 250 ng/mL and EGF at 50 ng/mL for 24 hours. The RNA was isolated from those cells and MMP-10 expression was analyzed by qRTPCR. Both HGF and EGF enhanced MMP-10 expression, with EGF inducing a more pronounced increase (Figure 19A,B). These experiments showed that MMP-10 can be up-regulated in AGS cells via c-Met and EGFR. Next the role of these two RTKs in the RESULTS 39 induction of MMP-10 during infection was addressed. To achieve this aim, AGS cells were treated with a Non-silencing siRNA or with siRNA directed to c-Met, and with a chemical inhibitor of EGFR, AG-1478, or with DMSO alone, and infected with H. pylori or left untreated for 24 hours. The downregulation of c-Met and EGFR abrogated H. pylorimediated MMP-10 expression (Figure 19C,D). These findings indicate that the increase in MMP-10 expression induced by H. pylori is mediated by the activation of c-Met and EGFR. Figure 19. H. pylori increases MMP-10 expression via EGFR and c-Met. AGS cells were treated with HGF at 250 ng/mL (A) and EGF at 50 ng/mL (B) for 24 hours. (C-D) AGS cells were infected with H. pylori strain 26695 for 24 hours at a MOI of 100, after the transient tranfection with a Non-silencing siRNA (NS) or an siRNA directed to c-Met (si c-Met) (C), or after treatment with the EGFR chemical inhibitor AG-1478 at a final concentration of 5µM (iEGFR), or with DMSO, 1 hour before the infection (D). (A-D) MMP-10 expression was analyzed by qRT-PCR. MMP-10 expression levels were normalized to GAPDH expression and results are presented as fold differences relative to uninfected cells (A-B), cells treated with a Non-silencing siRNA (C) or with DMSO (D). Data correspond to the mean values +/- SE and are representative of three independent experiments. *, significantly different from uninfected cells; **, significantly different from non-silenced cells or cells treated with DMSO infected with wild type H. pylori strain. RESULTS 40 2.6. Role of c-Met and EGFR downstream targets in H. pylori-induced MMP-10 expression In order to have a more clear picture of the molecules involved in c-Met and EGFR signaling that lead to MMP-10 up-regulation in H. pylori infection, and because in Part I.1 several c-Met downstream targets that also function downstream EGFR were found to be important for H. pylori-induced cell invasion, the involvement of Nck, PLC-γ, and c-Src in H. pylori-induced MMP-10 expression was addressed. To fulfill this aim, the expression of Nck, PLC-γ, and c-Src in AGS cells was downregulated using siRNAs, and cells were infected for 24 hours with H. pylori. As a control, AGS cells were transfected with a Non-silencing siRNA. Since the silencing of cSrc was difficult to achieve, the chemical inhibitor PP2 directed to the Src family kinases was also used. Cells were treated one hour before infection with PP2 or DMSO and then infected for 24 hours. RNA was isolated after this period and analyzed by qRT-PCR. As expected, MMP-10 expression increased in AGS cells infected with H. pylori, but this increase was attenuated in cells silenced for Nck (Figure 20A). Similarly H. pylori-induced MMP-10 expression was decreased in cells silenced for c-Src or in which c-Src activity was inhibited with PP2 (Figure 20C,D). In contrast, the silencing of PLC-γ had no effect in H. pylori-mediated MMP-10 expression (Figure 20B). These results point to a role for Nck and c-Src in the regulation of MMP-10 during infection. RESULTS 41 Figure 20. Nck and c-Src, but not PLC-γ, are involved in H. pylori-mediated MMP-10 expression (A-C) AGS cells alone or transiently transfected with a Non-silencing siRNA (NS) or with siRNAs directed to Nck, PLC-γ and c-Src, or (D) after a treatment for 1hour with the Src family kinases chemical inhibitor PP2 (iSrc), or DMSO, were infected with H. pylori strain 26695 for 24 hours at a MOI of 100. MMP-10 expression was analyzed by qRT-PCR. MMP-10 expression levels were normalized to GAPDH expression and results are presented as fold differences relative to cells treated with a Non-silencing siRNA (A-C) or with DMSO (D). Data correspond to the mean values +/- SE and are representative of three independent experiments. *, significantly different from uninfected cells; **, significantly different from non-silenced cells or cells trated with DMSO infected with a wild type H. pylori strain. 2.7. Analysis of the participation of ERK1/2, JNK and p38 signaling pathways in H. pylori-mediated MMP-10 expression Because ERK, JNK, and p38 pathways are involved both in the induction of different MMPs (Ridley et al., 1997, Reunanen et al., 1998) and in H. pylori-driven signaling (Naumann et al., 1999, Wessler et al., 2000), and as they can function downstream c-Met and EGFR (Terakado et al., 2011, Brusevold et al., 2012, Tsai et al., 2012), it was next determined if these pathways were involved in H. pylori-mediated MMP-10 expression. For this purpose, the effect of chemical inhibitors of ERK1/2 (U-0126), JNK (SP600125), and p38 (SB203580) on MMP-10 expression after infection was evaluated. AGS cells were treated with the inhibitors or with the vehicle (DMSO) alone, 1 hour before infection. Cells were then infected with H. pylori 26695 for 24 hours in the presence of the inhibitors, and MMP-10 expression was measured by qRT-PCR. Infection with H. pylori in the presence of DMSO did not affect MMP-10 expression stimulated by the bacteria alone (Figure 21). The treatment with the ERK1/2 inhibitor resulted in complete abrogation of MMP-10 expression and treatment with the JNK inhibitor led to a significant decrease of MMP-10 expression after H. pylori infection (Figure 21A). On the contrary, the inhibition of RESULTS 42 p38 resulted in enhanced expression of MMP-10 after infection (Figure 21B). Taken together, these results suggest that MMP-10 expression induced by H. pylori is partially mediated by JNK and ERK1/2 pathways, and that p38 may have an inhibitory role in this process. Figure 21. H. pylori increases MMP-10 expression through ERK and JNK pathways. AGS cells were treated 1 hour before the infection with DMSO or with chemical inhibitors of ERK1/2 (U-0126, 25 µM; iERK) (A), JNK (SP600125, 20 µM; iJNK) (A), and p38 (SB203580, 20 µM; ip38) (B). (A-B) After treatment AGS cells were infected with H. pylori 26695 for 24 hours at a MOI of 100 (Hp). MMP-10 expression was evaluated by qRTPCR. MMP-10 expression levels were normalized to GAPDH expression and results are presented as fold differences relative to uninfected cells treated with DMSO. Data correspond to the mean values ± SE and are representative of three independent experiments. *, significantly different from uninfected cells; **, significantly different from cells treated with DMSO infected with a wild type H. pylori strain. 2.8. Analysis of MMP-10 modulation by cytokines produced by H. pylori infection Several cytokines are able to modulate MMP secretion in pathological contexts such as chronic inflammatory diseases (Zenmyo et al., 1996). Since the infection with H. pylori induces chronic gastritis and it is known that pro-inflammatory cytokines are produced in response to H. pylori infection, it was next evaluated whether these cytokines play a role in the modulation of MMP-10 expression. A panel of proand anti-inflammatory cytokines available in the laboratory was used. AGS cells were treated for 24 hours with recombinant IL-1β, TNF-α, TGF-β, and IL-10, at a final concentration of 10 ng/mL, with IL-8 at 8 ng/mL, and with INF-γ at 100U/mL. RESULTS 43 After this period, RNA was isolated and MMP-10 expression was analyzed by qRT-PCR. The graph on Figure 22 shows that from the panel of cytokines tested, only IL-1β was able to induce a significant increase in MMP-10 expression. This suggests that not only H. pylori, but also IL-1β that is produced in the gastric mucosa in response to the infection may contribute to MMP-10 modulation. Figure 22. IL-1β treatment increase MMP-10 expression. AGS cells were treated with IL-1β, TNF-α, TGF-β and IL-10 at a final concentration of 10 ng/mL and, with IL-8 at 8 ng/mL and INF-γ at 100U/mL for 24 hours. MMP-10 expression was evaluated by qRT-PCR. MMP-10 expression levels were normalized to GAPDH expression and results are presented as fold differences relative to untrated cells. Data on graphs represents the mean value +/- SE and are representative of three independent experiments, except for IL-10 and IL-8 with two independent experiments. *, significantly different from non-treated cells. 2.9. Role of MMP-10 in MMP-1 activation in H. pylori infection Several MMPs are activated via proteolytic cleavage by other MMPs (Mannello et al.). MMP-10 can activate other MMPs, namely MMP-1 (Nakamura et al., 1998, Windsor et al., 1993), a metalloproteinase that is up-regulated by H. pylori, and is involved in gastric tissue degradation and remodeling (Krueger et al., 2006, Pillinger et al., 2007, Sokolova et al., 2012). Indeed, in our model system, MMP-1 was also found to be up-regulated in AGS cells by H. pylori infection (Figure 23). RESULTS 50 1.3. Role of E-cadherin in H. pylori-mediated cell invasion Giving that AGSEcad cells have a functional adherens junctions complex, it was next investigated whether E-cadherin was sufficient to inhibit H. pylori-mediated invasive phenotype. AGS, AGSEcad, and NCI-N87 cells were infected with H. pylori strains 26695 and 60190, and used in Matrigel invasion assays. After infection, AGSEcad cells displayed significantly lower levels of invasion than AGS cells (Figure 27). Similar results were obtained in infected NCI-N87 cells that endogenously express E-cadherin, suggesting that E-cadherin expression is sufficient to suppress H. pylori-mediated invasive phenotype. Figure 27. E-cadherin counteracts H. pylori-mediated cell invasive phenotype. AGS, AGSEcad, and NCI-N87 cells infected or not for 24 hours with H. pylori strain 26695 or 60190 on Matrigel-coated filters. Graphics represent the mean value of invasion +/- SD and are representative of three independent experiments. *, significantly different from uninfected cells; **, significantly different from AGS cells infected with H. pylori. 1.4. Role of E-cadherin in H. pylori-mediated c-Met and p120-catenin tyrosine phosphorylation Since the cell motogenic response and invasion induced by H. pylori in AGS cells require c-Met phosphorylation (Churin et al., 2003, Oliveira et al., 2006), the effect of E-cadherin on the phosphorylation status of c-Met was investigated. In addition and because p120catenin tyrosine phosphorylation is modulated by E-cadherin (Ozawa et al., 2001), the phosphorylation status of p120-catenin was also investigated. RESULTS 51 Lysates of H. pylori-infected AGS and AGSEcad cells were immunoprecipitated with an antibody recognizing tyrosine-phosphorylated residues, and immunoblotted with anti-cMet and anti-p120-catenin antibodies (Figure 28). The expression of c-Met and of p120-catenin was not altered by H. pylori in any of the cell lines. In AGS cells, H. pylori increased the phosphorylation level of c-Met and of p120catenin. In contrast, in AGSEcad cells H. pylori decreased the phosphorylation levels of both proteins. These results were further confirmed in the NCI-N87 cell line, where tyrosine phosphorylation levels of c-Met and p120-catenin decreased after infection (Figure 28). Overall, these results suggest that E-cadherin suppresses H. pylori-mediated phosphorylation of c-Met and of p120-catenin. Figure 28. E-cadherin counteracts H. pylori-mediated c-Met and p120-catenin tyrosine phosphorylation. Western blot analysis of AGS, AGSEcad and NCI-N87 cells infected with H. pylori for 1 hour. Total cell lysates were immunoprecipitated with an antibody against tyrosine-phosphorylated residues (PY-20) and immunostained with anti-c-Met or anti-p120-catenin antibodies. In parallel, total cell lysates were immunostained with the same antibodies to control differences of expression. α-tubulin immunostaining was used as loading control. Graphics represent the variation in c-Met and p120-catenin tyrosine phosphorylation in comparison to the endogenous phosphorylation levels of uninfected cells. Data correspond to the mean value +/- SD and are representative of three independent experiments. *, significantly different from uninfected cells. RESULTS 52 1.5. Effect of H. pylori on the localization of elements of E-cadherin-catenin complex Using confocal microscopy, the localization of the elements of the E-cadherin/catenin complex after H. pylori infection was analysed. One hour after infection with H. pylori, there was an increase in the intensity of membrane staining of E-cadherin and of p120catenin (Figure 29), and in the nuclear levels of β-catenin (data not shown). No differences regarding α-catenin were observed. Figure 29. H. pylori affects the localization of elements of the E-cadherin/catenin complex. AGSEcad monolayers were infected with H. pylori for 1 or 6 hours. After fixation, monolayers were stained with antibodies specific for E-cadherin, p120or α-catenins (green). Nuclei were counterstained with DAPI (red). Scale bar represents 40 µm. Images are representative of three independent experiments. Since CagA localizes to the inner surface of the plasma membrane after injection (Segal et al., 1999, Asahi et al., 2003), and the membrane staining of E-cadherin and p120catenin increased after infection (Figure 29), the putative co-localization between CagA and these E-cadherin/catenin complex elements was investigated. Analysis of the xz and yz confocal sections suggest that after 1 hour of infection of AGSEcad cells, CagA partially co-localizes with E-cadherin and with p120-catenin RESULTS 53 (arrowheads). Immunofluorescent signal corresponding to CagA was also visualized in areas negative for p120-catenin and E-cadherin (Figure 30). Specificity of the immunostaining was confirmed with non-immunizing IgGs of the same isotype. Figure 30. CagA, E-cadherin and p120-catenin co-localization during infection. AGSEcad monolayers infected with H. pylori for 1 hour were double immunostained for E-cadherin (green) and CagA (red) or p120catenin (green) and CagA (red). Immunostainings were also performed using a CagA isotype antibody as control. xz or yz sections for both channels or for each channel apart can be visualized in more detail below each panel. Arrowheads indicate areas of co-localization. Images were obtained with a confocal microscope and are representative of two independent experiments. Scale bar represents 10 µm. 1.6. Analysis of H. pylori CagA interactions with E-cadherin and with p120catenin Since H. pylori CagA interacts and co-localizes with elements of the E-cadherin/catenin complex, the role of H. pylori virulence protein CagA in this interaction was investigated. Lysates of H. pylori-infected AGSEcad cells were immunoprecipitated with anti-Ecadherin or anti-p120 catenin antibodies, and immunoblotted with an anti-CagA antibody. It was observed that CagA co-immunoprecipitated with E-cadherin and, interestingly, it was also observed that CagA co-immunoprecipitated with p120-catenin (Figure 31A). RESULTS 54 Immunoprecipitation with non-immunizing IgGs of the same isotype confirmed the specificity of such interactions. Likewise, in NCI-N87 cells CagA imunoprecipitated with Ecadherin and also with p120-catenin (Figure 31A). Furthermore, in both AGSEcad and NCI-N87 cells, in the presence of bacteria the binding between E-cadherin and p120catenin was enhanced (Figure 31B,C). No differences were observed regarding the βcatenin/α-catenin interaction. Thus, our results indicate that after infection, H. pylori CagA binds to elements of the E-cadherin/catenin complex. Figure 31. H. pylori CagA interacts with E-cadherin, with p120-catenin, and with c-Met. (A) AGSEcad and NCI-N87 cells were infected with H. pylori for 1 hour. Cell lysates were immunoprecipitated with anti-Ecadherin, anti-p120-catenin or anti-c-Met antibodies, and used for Western blot to be immunostained with an anti-CagA antibody. Immunoprecipitation was also performed with the same isotype control IgGs. Immunoblots were restained for E-cadherin or p120-catenin as loading controls. (B) AGSEcad cells were infected with H. pylori for 1 hour. Cell lysates were immunoprecipitated with anti-E-cadherin, anti-p120-catenin or anti-β-catenin antibodies, and used for Western blot to be immunostained with anti-p120-catenin, anti-Ecadherin, and anti-α-catenin antibodies. Immunoblots were restained for p120or β-catenins, as loading RESULTS 55 controls. (C) NCI-N87 cells were infected with H. pylori for 1 hour. Cell lysates were immunoprecipitated with anti-E-cadherin or anti-p120-catenin antibodies, and used for Western blot to be immunostained with the same antibodies. Immunoblot was restained for E-cadherin as loading control. Graphics represent the variation in protein co-immunoprecipitation in comparison to the endogenous levels of uninfected cells. Data correspond to the mean value +/- SD and are representative of three independent experiments. *, significantly different from uninfected cells. Since CagA targets the c-Met receptor, inducing its phosphorylation (Churin et al., 2003, Oliveira et al., 2006), and activating downstream effector molecules, it was next examined whether the interactions between CagA and E-cadherin, and CagA and p120-catenin occurred downstream c-Met. Co-immunoprecipitation studies on H. pylori-infected AGSEcad cells confirmed that CagA interacts with c-Met (Figure 31A) as previously described (Churin et al., 2003). Interestingly, interactions established between c-Met and E-cadherin, and between c-Met and p120-catenin were enhanced after infection with H. pylori 26695 and 60190 (Figure 32A). In agreement with these observations, in H. pylori-infected NCI-N87 cells, increased binding of c-Met and E-cadherin and of c-Met and p120-catenin was also observed (Figure 32A). When cells were infected with a cagA mutant strain (60190CagA - ), there was a decrease in c-Met/E-cadherin but not in c-Met/p120-catenin interactions (Figure 32B), suggesting that CagA plays a role in the formation of the c-Met/E-cadherin interaction. Studies with siRNA targeting c-Met did not affect the expression of p120-catenin and, as expected, abolished the interactions between c-Met and p120-catenin (Figure 32C) and between c-Met and E-cadherin (data not shown). Noteworthy, silencing of c-Met abolished the interactions between CagA and p120-catenin and also between CagA and E-cadherin (Figure 32D). These results suggest that interactions established between CagA and E-cadherin and between CagA and p120-catenin are c-Met dependent. RESULTS 56 Figure 32. The interaction established between CagA, E-cadherin, and p120-catenin is mediated by c-Met. (A) AGSEcad and NCI-N87 cells were infected with H. pylori for 1 hour and cell lysates were immunoprecipitated with anti-E-cadherin or anti-p120-catenin antibodies and used for Western blot to be immunostained with an anti-c-Met antibody. Anti-c-Met and antiα-tubulin antibodies were used as loading controls. (B) AGSEcad cells were infected with H. pylori strain 60190 and with its cagA mutant (60190CagA - ) for 1 hour. Cell lysates were immunoprecipitated with anti-E-cadherin or anti-p120-catenin antibodies and used for Western blot to be immunostained with an anti-c-Met antibody. Anti-c-Met and anti-α-tubulin antibodies were used as loading controls. (C) AGSEcad cells were transiently transfected with siRNA directed to c-Met. The effect of transfection on c-Met, p120-catenin and α-tubulin expression was evaluated by Western blot with specific antibodies. (D) AGSEcad and NCI-N87 cells transfected or not with c-Met siRNA were infected with H. pylori for 1 hour. Cell lysates were immunoprecipitated with anti-p120-catenin and anti-E-cadherin antibodies and RESULTS 57 used on Western blot to be immunostained with anti-c-Met, and anti-CagA antibodies. Data correspond to the mean value +/- SD and are representative of three independent experiments. *, significantly different from uninfected cells. Altogether, the results presented in Part II of the Results evidence that H. pylori alters the localization of elements of the E-cadherin/catenin complex, leading to formation of a multiproteic complex composed by CagA, c-Met, E-cadherin, and p120-catenin. The formation of this complex impairs c-Met and p120-catenin tyrosine phosphorylation and suppresses the cell invasive phenotype induced by H. pylori. DISCUSSION DISCUSSION 66 in MMP-10 modulation after infection. Giving that Nck is an adaptor protein of the RTKs studied, it may function in the early stages of signaling transduction. The role of c-Src is not surprising since this is a kinase of CagA, a virulence factor that is essential to MMP10 increased expression. Whether the phosphorylation of CagA is necessary to MMP-10 up-regulation will be an interesting issue to address. Indeed, in a previous report the inhibition of CagA phosphorylation using a c-Src chemical inhibitor resulted in a decrease of H. pylori-mediated MMP-9 secretion (Nam et al., 2011). Our Group has previously shown that H. pylori T4SS-competent and CagA-positive strains induce AGS cell invasion via c-Met receptor activation and increased activities of MMP-2 and MMP-9 (Oliveira et al., 2006). It is plausible that phosphorylation of tyrosine residues at the c-Met receptor intracellular domains mediated by CagA results in the phosphorylation and binding of adaptor proteins and activation of signal transducers, including Nck and c-Src, eventually leading to MMP-10 expression and cell invasion. Our Group has previously observed that the increase in the proteolytic activities of MMP2 and MMP-9 upon H. pylori infection, significantly decreased after silencing c-Met expression, suggesting that c-Met is involved in the activation of these MMPs (Oliveira et al., 2006). Adding to this observation, it has been shown that MMP-10 cleaves and activates pro-MMP-9 (Nakamura et al., 1998). Whether c-Met-mediated MMP-10 expression plays a role in pro-MMP-9 activation in the context of H. pylori infection will be an interesting topic of further studies. It was demonstrated that the ERK1/2 and JNK signaling pathways are involved in H. pylori-induced MMP-10 expression. These observations, together with the finding that EGFR is involved in H. pylori stimulation of MMP-10 are also consistent with previous reports of H. pylori-mediated ERK activation via transactivation of the EGFR (Keates et al., 2001, Keates et al., 2005). The involvement ERK1/2, JNK, and p38 signal transduction pathways in MMP regulation has been previously described (Reunanen et al., 1998, Ridley et al., 1997), including in the context of H. pylori infection (Crawford et al., 2003, Krueger et al., 2006, Pillinger et al., 2007, Nam et al., 2011). In contrast to ERK and JNK, our results showed that inhibition of p38 signaling significantly stimulated MMP10 expression. Curiously, the finding that inhibition of p38 enhanced MMP-10 expression was also reported in head and neck cancer (Deraz et al., 2011). Our results with H. pylori infection are similar to those obtained by Pillinger et al. using the same AGS cell line, and which detected a significantly enhancing effect of the p38 inhibitor SB203580 on MMP-1 expression (Pillinger et al., 2007). Similar findings were also obtained in AGS cells by Nam et al. in which p38 inhibitor slightly activated MMP-9 secretion (Nam et al., 2011). DISCUSSION 67 A panel of cytokines associated to H. pylori infection, including IL-1β, TNF-α, INF-γ, TGFβ, IL-10, and IL-8 (Romero-Adrian et al., 2010), were evaluated in the modulation of MMP-10 expression. Of these, only IL-1β was able to induce an overexpression of MMP10. This is particularly interesting giving the previously described role of IL-1β during H. pylori infection in increasing gastric cell secretion of MMP-3 (Gooz et al., 2003), a MMP of the same sub-family of MMP-10. The results obtained here suggest a role for MMP-10 during H. pylori infection in the activation of MMP-1. However, that is probably not the only mechanism of MMP-1 activation, since the silencing of MMP-10 was not sufficient to abrogate active MMP-1 present in conditioned medium. This is particularly interesting because MMPs are regulated at several levels, both transcriptional and post-transcriptional. One of the posttranscriptional regulatory events is the activation of the pro-enzyme to an active form, which is probably what occurs in MMP-1 regulation by MMP-10. While previous works that described an association between MMP-1 up-regulation and H. pylori infection have focused on the transcriptional regulation (Krueger et al., 2006, Pillinger et al., 2007, Sokolova et al., 2012), this is the first study describing a post-transcriptional process leading to MMP-1 activation. The fact that MMP-10 may function as activator of other MMPs, makes this metalloproteinase a special player in the amplification of the general MMP response during H. pylori infection. The studies associating the up-regulation of MMPs to infection with H. pylori usually describe the final result of MMP activation as effectors of invasion and migration. However, MMPs are also molecular regulators able to modulate a plethora of chemokines, reactive oxygen species, and growth factors that, in turn, act in the induction of several pathological processes (Parks et al., 2004). Future studies addressing the role of MMPs during H. pylori infection not only as modulators of tissue damage, but also as modulators of other pathological processes, should be of critical importance. The increase in production of MMPs associated to infection with H. pylori virulent strains may account for gastric tissue damage and for the ability of host epithelial cells to invade surrounding tissues. This may also suggest a role for the bacterium in later stages of gastric carcinogenesis. In conclusion, these results show that MMP-10 expression is induced in gastric epithelial cells by CagA-positive H. pylori strains via the c-Met and EGF receptors, in a process that involves Nck and Src, and also the ERK and JNK pathways. Our results also demonstrate that MMP-10 is involved in H. pylori-mediated gastric cell invasion (Figure 33). In the future, it will be interesting to address whether targeting MMPs in the context DISCUSSION 68 of H. pylori infection, either by direct inhibitors or by targeting the signaling pathways that up-regulate MMP expression, will improve the outcome of the infection. Figure 33. H. pylori-mediated cell invasion and MMP-10 up-regulation. H. pylori CagA-positive strains induce gastric cell invasion via c-Met-mediated PLC-γ, Nck, and c-Src activation. MMP-10 is up-regulated by H. pylori cagA-positive strains via the c-Met and EGFR, in a process that involves Nck and c-Src, and also ERK and JNK pathways. MMP-10 is also an important molecule for cell invasion induced by H. pylori. Green arrows refer to findings reported in this thesis regarding H. pylori-mediated cell invasion, and dark blue arrows refer to results regarding MMP-10 up-regulation. Part II. Analysis of the effects of E-cadherin in H. pylori-mediated cell invasion and signaling In Part II of the Results, it was demonstrated that H. pylori alters the E-cadherin/catenin complex, leading to the formation of a multiproteic complex composed of CagA, c-Met, Ecadherin, and p120-catenin. Due to the establishment of this complex, H. pylori-induced c-Met and p120-catenin phosphorylation as well as H. pylori-induced cell invasive phenotype were abrogated. In cell lines containing an intact E-cadherin/catenin complex, H. pylori induced lower DISCUSSION 69 levels or no invasion in comparison with AGS cells. Using two clones from an E-cadherin stably transduced cell line (AGSEcad), in which a functional E-cadherin/catenin complex was established, it was shown that E-cadherin was sufficient to suppress the invasive phenotype induced by H. pylori. Induction of AGS cell invasion by H. pylori involves c-Met tyrosine phosphorylation (Oliveira et al., 2006). In contrast, H. pylori decreases the phosphorylation levels of c-Met in cells with a stable E-cadherin/catenin complex. Also, depending on the E-cadherin status, infection with H. pylori differently affects the tyrosine phosphorylation of p120catenin. In cells lacking E-cadherin expression, H. pylori induced p120-catenin phosphorylation, whereas in cells expressing E-cadherin H. pylori led to reduced p120catenin phosphorylation. Taken together, these data point to a role of E-cadherin in the suppression of H. pylori-induced c-Met and p120-catenin phosphorylation and, consequently, in the suppression of the cell invasive phenotype. As already mentioned, c-Met is a receptor tyrosine kinase with a well-documented participation in cell invasion. p120-catenin interacts with the cytoplasmic juxtamembrane domain of E-cadherin (Yap et al., 1998), which, in conjunction with the phosphorylation status of p120-catenin, plays an important role in the stabilization of E-cadherin (Reynolds et al., 2004a, Yanagisawa et al., 2006). Phosphorylation of p120-catenin promotes its dissociation from E-cadherin and its translocation to the cytoplasm or nucleus where it participates in many signaling events (van Hengel et al., 1999). Endogenous p120catenin is described to promote migration and invasiveness of E-cadherin-deficient cells (Yanagisawa et al., 2006). In agreement with these findings are the ones performed in experimental models, where re-establishment of E-cadherin function in E-cadherindeficient cell lines reversed the invasive phenotype, pointing to E-cadherin role in suppression of cell invasion (Frixen et al., 1991, Vleminckx et al., 1991, Suriano et al., 2003a). The phosphorylation statuses of c-Met and of p120-catenin are regulated by kinases and phosphatases such as c-Src, Fer, Shp-2, and PTP1B (Reynolds et al., 1989, Kim et al., 1995, Balsamo et al., 1996, Ukropec et al., 2000). Because differences in cell invasion are associated with differences in c-Met and p120-catenin phosphorylation, a potential role for these kinases and phosphatases in the process of invasion is an interesting issue to explore. In both AGSEcad and NCI-N87 cells, E-cadherin expression levels were not altered by H. pylori, which is in accordance with previously published data in in vitro models (Conlin et al., 2004, Bebb et al., 2006). Although there is one description of an association between H. pylori infection and downregulation of E-cadherin expression (Terres et al., 1998), the DISCUSSION 70 findings presented in Part II of the Results of this thesis are also in accordance with those of the majority of the studies using gastric biopsy specimens that show no association between the two events (Bebb et al., 2006, Zullo et al., 2004, Shun et al., 2001, Chan et al., 2003b). In line with the observations of others, it was observed that H. pylori infection also did not affect the expression levels of p120-, β-, or α-catenins (Bebb et al., 2006, Krueger et al., 2007). Although no differences were observed in the expression of these molecules, H. pylori infection led to increased intensity in membrane staining of Ecadherin and of p120-catenin. These were also the experimental evidences from the immunoprecipitation studies, showing enhanced E-cadherin/p120-catenin binding after infection, suggesting that H. pylori alters the organization of the E-cadherin/catenin complex. After injection into the host cell cytoplasm the CagA T4SS effector localizes to the inner surface of the plasma membrane (Higashi et al., 2005). The confocal immunostainings pointed to co-localization of CagA and E-cadherin, and, most interestingly, to colocalization of CagA and p120-catenin at the cell membrane. The immunoprecipitation assays using in vitro infection revealed that CagA indeed physically interacts with Ecadherin, with c-Met, and with p120-catenin. The finding that CagA interacts with Ecadherin is in agreement with the work of Murata-Kamiya et al. who reported that in CagA-transfected cells, CagA interacts with E-cadherin, destabilizing the E-cadherin/βcatenin binding (Murata-Kamiya et al., 2007). The findings reported in this thesis also confirm previous observations of the interaction between CagA and c-Met (Churin et al., 2003), and add novelty to the field of study by showing interaction between CagA and p120-catenin. Another interesting observation in this study was that after H. pylori infection, c-Met/Ecadherin, and c-Met/p120-catenin interactions were enhanced. Taken together with the CagA interactions results, these observations suggest that CagA, E-cadherin, p120catenin, and c-Met interact with each other possibly forming a multiproteic complex. Furthermore, experiments with siRNA targeting c-Met revealed that, in the absence of cMet, CagA was no longer able to interact with E-cadherin or with p120-catenin, suggesting that interactions of CagA with the two elements of the E-cadherin/catenin complex occur via c-Met. The hypothesis formulated taking these results into consideration is that after infection, injected CagA interacts with c-Met and this complex now interacts with the Ecadherin/catenin complex, leading to the formation of a multiproteic complex (Figure 34). This model is supported by the co-immunoprecipitations and siRNA results and might explain the reorganization of the E-cadherin/catenin complex observed by DISCUSSION 71 immunocytochemistry. Further studies should be performed to elucidate how these four molecules interact within the multiproteic complex. There is now increasing evidence that E-cadherin participates in and regulates several signaling pathways via its extracellular domain (McLachlan et al., 2007). The extracellular domain of E-cadherin establishes not only homophilic, but also heterophilic interactions with tyrosine kinase receptors such as c-Met (Hiscox et al., 1999, Mateus et al., 2007, Reshetnikova et al., 2007). It is possible that the c-Met/E-cadherin interaction is established via their extracellular domain and that H. pylori CagA interacts with both E-cadherin and p120-catenin via c-Met. Figure 34. H. pylori and host cell invasion. (A) In cells with an intact E-cadherin/catenin complex H. pylori infection leads to enhanced c-Met/E-cadherin, c-Met/p120-catenin, and E-cadherin/p120-catenin interactions. In these cells, CagA binds to c-Met and this complex now interacts with the E-cadherin/catenin complex, suppressing H. pylori-mediated c-Met and p120-catenin phosphorylation and cell invasive phenotype (B) In Ecadherin negative cells, H. pylori CagA leads to an increase in the phosphorylation levels of both p120catenin and c-Met. The activation of c-Met and its downstream targets Nck, c-Src, and PLC-γ results in host cell invasion. Disruption of the adherens junctions by H. pylori has been shown in several reports using cell line models (Conlin et al., 2004, Weydig et al., 2007, Schirrmeister et al., 2009, Hoy et al., 2010). These alterations may involve E-cadherin shedding via ADAM-10 induction by H. pylori or via the secreted bacterial protease HtrA (Schirrmeister et al., 2009, Hoy et al., 2010). Furthermore, it has been shown that H. pylori induces translocation of E-cadherin from the cell membrane to intracellular location (Conlin et al., 2004, Weydig et al., 2007, Schirrmeister et al., 2009). In both cases, E-cadherin alterations induced by H. pylori DISCUSSION 72 result in phenotypic changes such as cell scattering and elongation, increased cell migration and invasion. The apparent discrepancies between these observations and the results obtained in this thesis may derive from differences in infection time and cell lines used. Here, the phosphorylation and co-immunoprecipitation assays were performed 1 hour after infection, a short-term infection time, and it is possible that longer infection periods result in different phenotypes. Nevertheless, the invasion assays were performed after 24 hours of infection, and corroborate the results obtained after 1 hour infection, showing that the potential inactivation of the c-Met due to its interaction with E-cadherin suppresses the invasive phenotype induced by the bacteria. It should also be noted that when one is addressing the issue of H. pylori infection in cellcell junction dysfunction two main aspects should be taken into consideration. First, a lot of studies have been performed in cell line models, often non-human and non-gastric, and frequently cancer-derived and with impaired cell-cell junctions. For example, the AGS and the MKN45 cell lines harbor mutations in the E-cadherin gene (Yokozaki, 2000). Also, genetic alterations in components of the Wnt signaling pathway are common in cell lines such as MKN7, MKN28 and HT29 (Yokozaki, 2000, Hsi et al., 1999). In addition, in host cell lines and bacteria co-culture models, infections cannot easily be maintained for long time periods, making these models more close to acute that to chronic infections. Second, results obtained in cell lines do not match those observed in the gastric mucosa of H. pylori-infected patients. In particular, gastric biopsy specimens of patients with and without H. pylori infection do not show any relationship between the infection and changes in the expression or distribution of E-cadherin, β-catenin, or p120-catenin. This does not mean that the phenomena observed in vitro do not occur in vivo, but may be explained if H. pylori-induced alterations in the adherens junctions are fast and transient and, therefore, not observable in fixed gastric tissues. Another possible explanation is that alterations in adherens junctions induced by H. pylori only occur in a subset of cells. These cells may then be selected as having growth advantages or increased resistance to apoptosis, therefore rendering them more susceptible to accumulation of DNA lesions which may lead to malignant transformation. Concluding remarks It is possible to make an analogy between what occurs during infection with H. pylori and the exploitation of the natural resources by the human being. In order to obtain cheap DISCUSSION 73 food and raw materials to overcome its needs, Man can damage its ecosystem in an irreversible way, which can compromise the future viability of the species in the habitat. In a similar way, H. pylori infection, since it is chronic and acquired during childhood persisting through the host’s lifetime, may induce, in the long term, such a pressure in the ecosystem that compromises either the viability of the gastric tissue, or bacterial survival. Since we are talking about an ecosystem, it is important to stress that changes in one particular process may affect all the other processes of the system in a way that sometimes may not be very clear. Therefore, any particular molecular process should be understood as being part of something bigger, and the final phenotype observed may be understood as the interplay of several variables. It is known that host, bacterium, and environmental factors account for the differences in the clinical outcome of H. pylori infection. Although it is known that only a small proportion of infected individuals develop gastric carcinoma, the exact mechanisms underlying disease development mediated by H. pylori infection remain unraveled. The interaction between H. pylori and gastric epithelial cells leads to activation of host signaling pathways, modification of cellular functions, and induction of cell phenotypes important for carcinogenesis. Results in this thesis suggest that in cells with intact adherens junctions H. pylori leads to formation of a multiproteic complex composed of CagA, c-Met, E-cadherin, and p120catenin which impairs c-Met and p120-catenin tyrosine phosphorylation and suppresses cell invasion induced by H. pylori. In contrast, in the absence of E-cadherin, H. pylori infection associates with an increased ability of the cells to invade. It is arguable whether the invasive phenotype observed in the Matrigel in vitro cell model overlaps with tumor cell invasion, as observed during tumor progression in vivo. The latter is the result of a multistage process involving other events in addition to E-cadherin impairment; in fact it is arguable whether H. pylori would play a role in tumor invasion, because often, H. pylori is not present in full-blown gastric carcinoma. 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Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 6, 639-642. SUMMARY 98 Since the E-cadherin-catenin complex functions as an invasion suppressor, the next aim was to evaluate whether the complex had a role in H. pylori-mediated cell invasion and signaling. For that, gastric cell lines with intact E-cadherin-catenin complex were used. Also, and for the purpose of comparison with previous data obtained in the AGS background, an AGS cell line with a wild-type E-cadherin was established. For the characterization of the structure and function of the adherens junctions, immunofluorescence, confocal microscopy and aggregation assays were used. The phosphorylation and interactions between proteins of the adherens junctions were evaluated by co-immunoprecipitation and western blot. The invasion capacity of the different cell lines was assessed with Matrigel assays. Results presented in this part of the thesis evidence that H. pylori infection leads to formation of a multiproteic complex composed by CagA, c-Met, E-cadherin, and p120-catenin. This complex impairs H. pylorimediated c-Met and p120-catenin tyrosine phosphorylation and suppresses the invasive phenotype induced by H. pylori. Altogether, these results suggest that the interaction between H. pylori and gastric epithelial cells leads to activation of host signaling pathways, modification of cellular functions, and induction of cell phenotypes which underlie disease, namely gastric carcinoma. The elucidation of H. pylori-host interactions may provide further insights on H. pylori pathogenesis and on the mechanisms relevant to gastric disease development. S UMÁR I O SUMÁRIO 101 H. pylori é uma bactéria gram-negativa que infecta mais de metade da população humana, causando gastrite superficial crónica que pode progredir para doenças mais graves, incluindo o carcinoma gástrico. H. pylori induz alterações nas vias de sinalização da célula do hospedeiro que resultam em alterações no comportamento normal da célula. Um dos fenótipos menos explorados associados com a infecção por H. pylori é a invasão celular, um fenótipo, que envolve uma multiplicidade de processos, incluindo a migração celular e a degradação da matriz extracelular por MMPs (Metaloproteases da Matriz). O objectivo geral do trabalho apresentado nesta tese foi o de avaliar o papel da infecção por H. pylori na indução de invasão das células epiteliais gástricas. A abordagem inicial consistiu na avaliação da capacidade invasiva através de componentes da matriz extracelular de células AGS, uma linha celular gástrica humana não-invasiva, após a infecção com H. pylori. O papel do fatores de virulência bacterianos CagA, T4SS (Sistema de Secreção Tipo 4) e VacA também foi avaliado. Depois de estabelecer que o receptor c-Met era importante para invasão celular das células AGS, mediada por H. pylori, o papel dos alvos a jusante do c-Met foi estudado. Os resultados mostraram que as estirpes de H. pylori com um T4SS competente induzem invasão de células gástricas do hospedeiro através dos componentes da matriz extracelular, por uma via dependente da activação de c-Met, Nck, PLC-γ, e c-Src. Com base nos resultados acima, que apontam para um papel de H. pylori na indução de degradação da matriz extracelular das células do hospedeiro, e uma vez que as MMPs são importantes para a degradação da matriz extracelular, o objectivo seguinte foi o de avaliar o papel de H. pylori na expressão de MMPs. O estudo começou com a validação de um painel de MMPs sobre-expressas num microarray de expressão de cDNA realizado pelo Grupo. Após a confirmação de que a infecção por H. pylori aumentou a expressão das MMP-1, MMP-7 e MMP-10, foi investigado mais detalhadamente o efeito de H. pylori na modulação da MMP-10. Em células não infectadas e infectadas por H. pylori foram feitos ensaios de real-time-PCR, western blot, ensaios de actividade enzimática e invasão. O papel dos factores de virulência CagA e T4SS de H. pylori foi estudado utilizando estirpes mutantes e um painel de isolados clínicos de H. pylori, com o status do cagA conhecido. O envolvimento de vias de sinalização celular do hospedeiro e de receptores tirosina-cinase na modulação da expressão de MMP-10 por H. pylori foi avaliado em experiências com siRNA e com inibidores químicos. Os resultados obtidos na Parte I.2 dos Resultados mostraram que SUMÁRIO 102 a expressão de MMP-10 é induzida em células epiteliais gástricas por estirpes de H. pylori CagA-positivas, através dos receptores c-Met e EGFR, num processo que envolve as proteínas Nck e c-Src e também as vias de sinalização ERK e JNK, bem como o seu envolvimento na invasão celular mediada por H. pylori. Uma vez que o complexo E-caderina-cateninas funciona como supressor de invasão, o objectivo seguinte foi o de avaliar o papel do complexo na invasão e sinalização celulares mediada por H. pylori. Para isso, foram utilizados linhas celulares gástricas com o complexo E-caderina-cateninas intacto. Além disso, e para efeitos de comparação com os dados anteriores obtidos com as células AGS, foi estabelecida uma linha celular AGS com expressão de E-caderina de tipo selvagem. Para a caracterização da estrutura e função das junções de adesão foi utilizada imunofluorescência, microscopia confocal e ensaios de agregação. A fosforilação e as interacções entre proteínas das junções de adesão foram avaliados por coimunoprecipitação e western blot. A capacidade de invasão das diferentes linhas celulares foi avaliada com ensaios de Matrigel. Os resultados apresentados nesta parte da tese mostraram que a infecção por H. pylori leva à formação de um complexo multiproteico composto por CagA, c-Met, E-caderina e catenina p120 . Este complexo impede a fosforilação de residuos tirosina no c-Met e catenina p120 mediada por H. pylori e suprime o fenótipo invasivo induzido pela bacteria. No seu conjunto, estes resultados sugerem que a interacção entre H. pylori e as células epiteliais gástricas conduz à activação de vias de sinalização do hospedeiro, à modificação de funções, e à indução de fenótipos celulares que poderão estar subjacentes ao desenvolvimento de doença, nomeadamente de carcinoma gástrico. A elucidação das interações H. pylori-hospedeiro pode fornecer informações sobre a patogénese de H. pylori, e sobre possíveis mecanismos relevantes para o desenvolvimento de doenças gástricas associadas à infecção. P APERS Paper I Helicobacter pylori CagA activates matrix metalloproteinase-10 in gastric epithelial cells through ERK and JNK-mediated pathways Costa AM, Ferreira RM, Oliveira MJ, Carneiro F, Leite M, Figueiredo C. In preparation 1 Helicobacter pylori CagA activates matrix metalloproteinase-10 in gastric epithelial cells through ERK and JNK-mediated pathways Angela Margarida Costa 1,2 , Rui Manuel Ferreira 1,2 , Maria Jose Oliveira 3 , Fatima Carneiro 1,2,4 , Marina Leite 1 , and Ceu Figueiredo 1,2 1 Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP); 2 Department of Pathology and Oncology, Faculty of Medicine of the University of Porto; 3 Institute of Biomedical Engineering (INEB); 4 Department of Pathology, Centro Hospitalar S. João, Porto, Portugal Running Title: MMP-10 expression in response to H. pylori