Le laboratoire a établi un modèle de souris α6ΔIEC qui développe une inflammation chronique intestinale associée à la formation d’adénocarcinomes colorectaux. Ce modèle correspond à une délétion ciblée à l’épithélium intestinal de l’intégrine α6β4. Mon projet de thèse a consisté à définir les mécanismes qui influencent la transformation de lésions inflammatoires en adénocarcinomes. La caractérisation du modèle α6ΔIEC a permis de mettre en évidence plusieurs altérations : détachement de l’épithélium, régénération du tissu, prolifération, augmentation de la perméabilité intestinale, hypersécrétion du mucus, ségrégation anormale des bactéries, inflammation chronique et formation de tumeurs.Pour étudier la séquence et la cinétique des mécanismes, j’ai développé un modèle de souris inductible (α6ΔIECTAM). Cette lignée présente, deux semaines après l’invalidation de l’intégrine α6,les mêmes signes d’inflammation que les souris α6ΔIEC. Mon approche a consisté à dissocier les processus impliqués dans chacune des étapes-clés de la pathologie afin de définir la contribution respective de l’infection par les bactéries et du stress mécanique. / We generated a new mouse model, α6ΔIEC, in which the genetic ablation of α6 integrin from intestinal epithelial cells triggered the development of spontaneous colitis and colorectal cancer. My main goal was to define the mechanisms by which inflamed lesions degenerate into infiltrating adenocarcinomas. Loss of α6 integrin in this model resulted in epithelial barrier damage, enhanced permeability, altered mucus layers, abnormal bacterial segregation, chronic inflammation and tumor development.In order to define the sequence of events and the mechanisms involved at each stage of the disease, from inflamed to tumor lesions, I developed an inducible mouse model, α6ΔIECTAM, in which α6 integrin ablation was induced by tamoxifen treatment. This line recapitulates all aspects of inflammation observed in the α6ΔIEC model, as early as two weeks after tamoxifen treatment. In particular, I tried to define the respective contribution of infection by bacteria and mechanical stress during disease progression.

Integrins are a family of conserved cell surface receptors found throughout the animal kingdom. They comprise 24 dimers in mammals, and regulate a number of processes including cell survival, differentiation, and migration. These complex cellular responses involve processes such as cell attachment, spreading, and various signaling pathways, which in turn depend on the composition of the extracellular environment, on its mechanical properties, and involved integrin types. This thesis focuses on identifying molecules that signal downstream of integrins and how integrin-induced signals may differ dependent on the type of mechanical stimulus that is given. In Paper I, we show that cell spreading and the activation of AKT is regulated by the catalytic PI3K isoform p110α. An intact β1 integrin cytoplasmic tail and actin polymerization was needed for spreading, whereas the presence of FAK or SRC, or the interaction between p110α and RAS was dispensable. Paper II reports that the RICTOR-mTOR complex (TORC2) acts as the kinase downstream of β1 integrins in order to phosphorylate AKT on Ser473, which was functionally linked to cell survival. β1 integrins activated both AKT1 and AKT2, but seemed to prefer AKT2. The investigation of several receptor types with regard to their requirement of TORC2, PAK, and ILK for AKT Ser473 phosphorylation revealed that different kinds of receptors engage specific enzyme combinations depending on cell type and context. In the third paper, we demonstrate that adhesion- and mechanical stretch-induced integrin signaling lead to divergent protein phosphorylation patterns, and that most signals from cell adhesion were not dependent on intracellular contractility. This indicates that integrin ligand binding and mechanical stretch induce signaling via distinct mechanisms. Reactive oxygen species (ROS) derived from different cellular sources modulated these responses. Stretching primarily induced phosphorylation of ERK1/2, and this signal was markedly increased by a derivative of the antioxidant ascorbate and extracellularly administered catalase. The robust AKT phosphorylation in response to adhesion was almost completely abolished with an inhibitor targeting mitochondrial ROS, whereas phosphorylation levels were only marginally affected in stretch assays. Similar results were obtained with siRNA knock-down of a critical subunit of ROS-producing NADPH oxidases.

Neutrophils are implicated in many inflammatory lung disorders. However, the mechanisms regulating neutrophil migration in acute lung inflammation are incompletely understood. Although, integrin β2 mediates neutrophil migration in lungs in response to many stimuli such as E. coli, integrin involved in <i>S. pneumoniae</i> induced neutrophil migration is not known. Therefore, the role of integrin αvβ3 in neutrophil recruitment was tested. First, it was found that the number of neutrophils expressing the integrin subunits αv and β3 is reduced or remains in lung inflammation induced by E. coli or <i>S. pneumoniae</i>, respectively. Next, the role of integrin αvβ3 using β3 knockout mice (β3-/-) and function blocking antibodies was addressed. Neutrophil recruitment did not vary between wild type and β3-/- mice. Although β3 antibodies reduced neutrophil recruitment, similar effect was observed with isotype antibodies. Therefore, one can conclude that integrin αvβ3 is not critical for neutrophil recruitment in <i>S. pneumoniae</i> induced pneumonia. <p>Apart from integrins, TLR4 also regulate neutrophil migration. Because, the pattern of TLR4 expression at various times of lung inflammation is not known, TLR4 expression during different phases of lung inflammation in a rat model of LPS-induced inflammation was studied. TLR4 expression in the septum increased and decreased at 6h and 12-36h of inflammation, respectively. Since these correlate with the time of increase and decline of neutrophil recruitment, the findings support previously observed requirement for TLR4 in neutrophil recruitment. <p>Neutrophils recruited into the lungs regulate the inflammatory process by controlling subsequent monocyte/macrophage recruitment. The mechanisms involved and the pattern of monocyte/macrophage recruitment in lungs are not completely understood. Therefore, the possible involvement of monocyte chemoattractant protein (MCP)-1, which is a premier chemokine in monocyte/macrophage migration and produced by neutrophils and other cells was tested. This was addressed by quantification of monocytes/macrophages at various times and using neutrophil depletion experiments in LPS-induced lung inflammation in rats. It was found that monocytes/macrophages migrate very early and before neutrophils in addition to their migration in the late phase of acute lung inflammation. Neutrophil depletion abrogated both early as well as the late monocyte/macrophage recruitment without altering the expression of MCP-1. Therefore, possibly other chemokines and not MCP-1 are involved in neutrophil dependent monocyte/macrophage recruitment. <p>To conclude, the experiments further the understanding on acute lung inflammation by ruling-out the involvement of integrin αvβ3 and MCP-1 in β2-independent neutrophil migration and neutrophil dependent monocyte/macrophage recruitment, respectively. Further studies are essential to find the integrins and chemokines operating in the above situations. Equally important will be to understand the functional significance of early recruited monocytes/macrophages in the lung.

Neutrophils are implicated in many inflammatory lung disorders. However, the mechanisms regulating neutrophil migration in acute lung inflammation are incompletely understood. Although, integrin β2 mediates neutrophil migration in lungs in response to many stimuli such as E. coli, integrin involved in <i>S. pneumoniae</i> induced neutrophil migration is not known. Therefore, the role of integrin αvβ3 in neutrophil recruitment was tested. First, it was found that the number of neutrophils expressing the integrin subunits αv and β3 is reduced or remains in lung inflammation induced by E. coli or <i>S. pneumoniae</i>, respectively. Next, the role of integrin αvβ3 using β3 knockout mice (β3-/-) and function blocking antibodies was addressed. Neutrophil recruitment did not vary between wild type and β3-/- mice. Although β3 antibodies reduced neutrophil recruitment, similar effect was observed with isotype antibodies. Therefore, one can conclude that integrin αvβ3 is not critical for neutrophil recruitment in <i>S. pneumoniae</i> induced pneumonia. <p>Apart from integrins, TLR4 also regulate neutrophil migration. Because, the pattern of TLR4 expression at various times of lung inflammation is not known, TLR4 expression during different phases of lung inflammation in a rat model of LPS-induced inflammation was studied. TLR4 expression in the septum increased and decreased at 6h and 12-36h of inflammation, respectively. Since these correlate with the time of increase and decline of neutrophil recruitment, the findings support previously observed requirement for TLR4 in neutrophil recruitment. <p>Neutrophils recruited into the lungs regulate the inflammatory process by controlling subsequent monocyte/macrophage recruitment. The mechanisms involved and the pattern of monocyte/macrophage recruitment in lungs are not completely understood. Therefore, the possible involvement of monocyte chemoattractant protein (MCP)-1, which is a premier chemokine in monocyte/macrophage migration and produced by neutrophils and other cells was tested. This was addressed by quantification of monocytes/macrophages at various times and using neutrophil depletion experiments in LPS-induced lung inflammation in rats. It was found that monocytes/macrophages migrate very early and before neutrophils in addition to their migration in the late phase of acute lung inflammation. Neutrophil depletion abrogated both early as well as the late monocyte/macrophage recruitment without altering the expression of MCP-1. Therefore, possibly other chemokines and not MCP-1 are involved in neutrophil dependent monocyte/macrophage recruitment. <p>To conclude, the experiments further the understanding on acute lung inflammation by ruling-out the involvement of integrin αvβ3 and MCP-1 in β2-independent neutrophil migration and neutrophil dependent monocyte/macrophage recruitment, respectively. Further studies are essential to find the integrins and chemokines operating in the above situations. Equally important will be to understand the functional significance of early recruited monocytes/macrophages in the lung.

Cell adhesion to proteins adsorbed onto implanted surfaces is particularly important to host responses in biomedical and tissue engineering applications. Biomaterial surface properties influence the type, quantity and functional presentation (activity) of proteins adsorbed upon contact with physiological fluids, and modulate subsequent cell response. Cell adhesion to extracellular matrix proteins (e.g. fibronectin) is primarily mediated by the integrin family of cell-surface receptors. Integrins not only anchor cells, supporting cell spreading and migration, but also trigger signals that regulate survival, proliferation and differentiation. A fundamental understanding of the adhesive interactions at the biomaterial interface is critical to the rational design of biomaterial surfaces. Using model surfaces of self-assembled monolayers of alkanethiols on gold presenting well-defined surface chemistries (CH3, OH, COOH, NH2), we investigated the effects of surface chemistry on osteoblastic differentiation. We report that surface chemistry effectively modulates fibronectin adsorption, integrin binding, focal adhesion assembly and signaling to direct the osteoblast cellular functions of adhesion strength, gene expression and matrix mineralization. Specifically, surfaces presenting OH and NH2 functionalities provide enhanced functional presentation of adsorbed fibronectin, promoting specificity of integrin binding as well as elevating focal adhesion assembly and signaling. Furthermore, the OH and NH2 surfaces supported elevated levels of osteoblast differentiation as evidenced by osteoblast-specific gene expression and matrix mineralization. These results contribute to the development of design principles for the engineering of surfaces that direct cell adhesion for biomedical and tissue engineering applications. In particular, the understanding provided by this analysis may be useful in the engineering of surface properties for bone tissue repair and regeneration.

Progress towards the design and the application of small molecules as inhibitors of angiogenesis is reported. First, the regulation of hypoxia inducible transcription with epipolythiodioxopiperazine (ETP) natural products is discussed, beginning with the exploration of the physical and chemical properties of ETP skeletal analogs, xylylene-linked bis-diketopiperazines (1,4-piperazine-2,5-diones, DKPs).The design, synthesis and solid-state structures of a new class of xylylene-linked bis(1,4-piperazine-2,5-diones) are reported in an effort to extend the molecular framework of piperazine-2,5-diones. These compounds were derived from piperazine-2,5-dione as the core structure, synthesized via a new efficient route, and their crystal structures were determined. We examined the effects of side chain substitution on conformations of the linked bis-DKPs in the solid state. The results suggested that the interplay between the attractive intermolecular interactions and repulsive steric interactions of the substituents at the C6 and C6' positions of the diketopiperazine rings is important in determining the solid-state conformations of xylylene-linked bis(piperazine-2,5-diones).Asymmetric alpha-sulfenylation reactions were designed and performed as a potential route to the synthesis of epipolythiodioxopiperazine natural products. First, a chiral auxiliary approach is reported, sulfenylating chiral azomethines of alpha-amino acids as epipolythiodiketopiperazine precursors in yields of 55% with de as high as 74%. Asymmetric organocatalytic alpha-sulfenylation of substituted piperazine-2,5-diones is also reported, with chiral cinchona alkaloids as bases and benzyl-substituted electrophilic sulfur transfer reagents. The reaction was investigated with varied catalyst loading, type of sulfenylating agent, temperature and solvent. The effects of ring substitution and type of catalyst on yield and enantioselectivity of the reaction are reported. The synthetic utility of the asymmetric alpha-sulfenylation in context of the synthesis of epipolythiodioxopiperazine fungal metabolites is discussed.Finally, chemical approach towards the inhibition of angiogenesis by targeting alpha v beta 3 integrin antagonists with synthetic multifunctional boron neutron capture therapy (BNCT) integrin ligands is presented. The novel synthesis of an alpha v beta 3 integrin antagonist containing a free amine group for peripheral modification is reported, along with the preparation of a bifunctional BNCT integrin ligand and a trifunctional BNCT integrin ligand containing a fluorescent dye. Synthetic challenges and potential therapeutic applications of these ligands are discussed.

For cancer cells to initiate cell migration and progress to metastasize, epithelial genes must be silenced and the expression of mesenchymal genes must be upregulated. During prostate carcinogenesis, E-cadherin expression is downregulated through multiple mechanisms, the majority of which combine to silence E-cadherin expression through transcriptional regulation at the level of the E-cadherin promoter. Recently it has been discovered that there is transcriptional upregulation of the mesenchymal cadherin, N-cadherin during prostate cancer metastasis. Although N-cadherin expression can be detected in human prostate cancer and in prostate carcinoma cell lines, the mechanisms controlling the transcriptional regulation of N-cadherin in cancer are uncharacterized. This body of work offers the first evidence for the mechanisms controlling the transcriptional upregulation of N-cadherin expression in prostate carcinoma. We utilized anchorage independent culture to induce downregulation of N-cadherin expression, and then analyzed the necessary events for N-cadherin upregulation when cells attached to Fibronetin (FN). In order to determine the functional regions of the N-cadherin proximal promoter that were involved in the upregulation of N-cadherin expression, we cloned regions of the human N-cadherin 5’ proximal promoter, and regions of the first intron of the N-cadherin gene into a luciferase reporter vector. It was determined that the bHLH transcription factor Twist1 controlled the upregulation of N-cadherin transcription in PC-3 cells, through β1 integrin dependent nuclear localization of Twist1. A cis-element located in the first intron of the N-cadherin gene was shown to be necessary for Twist1 mediated effects on the N-cadherin promoter. We then determined the requirements for cell-type specific expression of the N-cadherin promoter. It was determined that an additional cis-element located in the first intron of the N-cadherin gene was necessary to repress N-cadherin promoter activity in cells lacking N-cadherin. Through deletion analysis of the N-cadherin promoter luciferase construct, a DNA binding site for the transcription factor FoxP1 was discovered. FoxP1 binds to the repressive cis-element in vitro, and mutation of the FoxP1 DNA binding site eliminated cell-type specific activity of the N-cadherin promoter. Therefore, we have documented that the aberrant expression of N-cadherin in prostate carcinoma involves alterations in both positive and negative transcriptional regulators.

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder which results from fetal platelet destruction by maternal antibodies against platelet antigens, including GPIIbIIIa (αIIbβ3 integrin) and GPIbα. β3 integrin is also expressed by angiogenic endothelial cells (ECs) and is required for angiogenesis. Therefore, we investigated whether anti-β3 antibodies in FNIT cross-react with blood vessels of the fetus/neonate and contribute to pathogenesis. Antibodies to GPIbα were used as controls. To mimic human FNIT, β3 integrin- or GPIbα-deficient female mice were immunized with wild-type platelets and bred with wild-type male mice. Pups in both groups had thrombocytopenia but intracranial hemorrhage was only observed in anti-β3-mediated FNIT. Anti-β3-mediated FNIT pups had increased apoptosis in the brain and impaired vascularization of the brain and retina. In addition, anti-β3 sera inhibited proliferation and vascular-like tube formation by ECs in vitro. Therefore, anti-β3 antibodies in FNIT likely impair angiogenesis in the developing fetus/neonate.

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder which results from fetal platelet destruction by maternal antibodies against platelet antigens, including GPIIbIIIa (αIIbβ3 integrin) and GPIbα. β3 integrin is also expressed by angiogenic endothelial cells (ECs) and is required for angiogenesis. Therefore, we investigated whether anti-β3 antibodies in FNIT cross-react with blood vessels of the fetus/neonate and contribute to pathogenesis. Antibodies to GPIbα were used as controls. To mimic human FNIT, β3 integrin- or GPIbα-deficient female mice were immunized with wild-type platelets and bred with wild-type male mice. Pups in both groups had thrombocytopenia but intracranial hemorrhage was only observed in anti-β3-mediated FNIT. Anti-β3-mediated FNIT pups had increased apoptosis in the brain and impaired vascularization of the brain and retina. In addition, anti-β3 sera inhibited proliferation and vascular-like tube formation by ECs in vitro. Therefore, anti-β3 antibodies in FNIT likely impair angiogenesis in the developing fetus/neonate.

Endocytic trafficking of platelet surface receptors plays a role in the accumulation of granule cargo (i.e. fibrinogen and VEGF) and thus could contribute to hemostasis, angiogenesis, or inflammation. However, the mechanisms of platelet endocytosis are poorly understood. The small GTP-binding protein, ADP-ribosylation factor 6 (Arf6), regulates integrin trafficking in nucleated cells; therefore, we posited that Arf6 functions similarly in platelets. To address this, we generated platelet-specific, Arf6 knockout mice. Arf6-/- platelets had a storage defect for fibrinogen but not other cargo, implying Arf6’s role in integrin αIIbβ3 trafficking. Additionally, platelets from Arf6-/- mice injected with biotinylated-fibrinogen, showed lower accumulation of the modified protein than did WT mice. Resting and activated αIIbβ3 levels, measured by FACS, were unchanged in Arf6-/- platelets. Arf6-/- platelets had normal agonist-induced aggregation and ATP release; however, they showed faster clot retraction and enhanced spreading, which appears due to altered αIIbβ3 trafficking since myosin light chain phosphorylation and Rac1 activation, in response to thrombin, were unaffected. Arf6-/- mice showed no hemostasis defect in tail-bleeding or FeCl3–induced carotid injury assays. These data suggest a role for Arf6 in integrin αIIbβ3 trafficking in platelets. Additionally, the regulation of Arf6 in platelets was also investigated, focusing on integrin αIIbβ3 outside-in signaling which was suggested to be responsible for the second wave of Arf6-GTP loss. G protein-coupled receptor kinase-interacting protein 1 (GIT1), a GTPase-activating protein (GAP) toward Arf6, is suggested to be involved in αIIbβ3 downstream signaling. I found that GIT1, complex with β-PIX, was translocated to the detergent-insoluble pellet upon human platelet activation, a process that is blocked by RGDS and myrArf6 peptide treatment. Moreover, tyrosine-phosphorylation of GIT1 was impaired by treatment with both peptides or with actin polymerization inhibitors. GIT1’s role in platelets was further studied using platelet-specific, GIT1 knockout mice. GIT1-/- platelets failed to show any defect, including clot retraction or fibrinogen storage. Unlike human platelets, GIT1 expression levels were much lower in mouse platelets, suggesting that GIT2 may be the functionally relevant Arf6-GAP in mouse platelets. The data in this dissertation identify that Arf6 mediates fibrinogen storage, implying its role in integrin αIIbβ3 trafficking in platelets.