Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Apart from its primary function in the synaptic hydrolysis of acetylcholine, acetylcholinesterase (AChE) has been shown through in vitro demonstrations to be able to promote various non-cholinergic functions, including cell adhesion and neurite outgrowth, differentiation, and amyloidosis. AChE was also shown to bind to mouse laminin-111 in vitro by an electrostatic mechanism. Previous results suggest that the site on AChE recognised by certain monoclonal antibodies (MAbs) might be critical for differentiation. These MAbs were found to inhibit both laminin binding and cell adhesion in neuroblastoma cells. In this study, the structure and characteristics of this site were investigated, using the AChE-laminin interaction as a template as well as a detailed epitope analysis of the MAbs. The interaction sites of AChE and laminin were investigated using phage display, modelling and docking, synthetic peptides, enzyme linked immunosorbent assays (ELISAs) and conformational interaction site mapping. Docking of AChE with the single-chain variable fragments (scFvs) produced from the phage display showed the major recognition motifs to be the 90Arg-Glu-Leu-Ser-Glu-Asp motif, the 40Pro-Pro-Met-Gly sequence, and the 59Val-Val-Asp-Ala-Thr-Thr (human) motif. Mouse AChE was found to interact with the basic structures Val2718-Arg-Lys-Arg- Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg-Lys2793; and Val2817-Glu-Arg-Lys2820, on the 1 G4 domain of laminin. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps with laminin’s heparin-binding site. Docking showed the major component of the interaction site on AChE to be the acidic Arg90-Glu-Leu-Ser-Glu-Asp95 (omega loop), and also involving the Pro40-Pro-Val42, Arg46 (linked to Glu94 by a salt bridge) and the hexapeptide Asp61 Ala-Thr-Thr-Phe-Gln66. Epitope analysis showed the MAb’s major recognition site to be the sequence Pro40-Pro- Met-Gly-Pro-Arg-Arg-Phe48 (human AChE). The MAbs also reacted with the prolinerich sequences Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 and Pro88-Asn-Arg-Glu-Leu-Ser-Glu- Asp95. These results define the interaction sites involved in the AChE-laminin interaction and suggest that the interaction plays a role in cell adhesion. Despite the in vitro demonstrations of the importance of AChE’s non-classical functions, the AChE knockout survives. Results from this study suggest the possibility of functional redundancy between AChE and other molecules in early development. Using these in vitro findings that AChE is able to bind laminin-111, information on the interaction sites, as well as results from the monoclonal antibody (MAb) epitope analysis, the idea of redundancy was investigated. Docking and bioinformatics techniques were used to investigate structurally similar molecules that have comparable spatiotemporal expression patterns in the embryonic nervous system. AChE has been shown to be involved in the pathogenesis of Alzheimer’s disease, thus molecules associated with brain function and neurodegeneration were also investigated. Molecules with which AChE could be possibly redundant are syndecans, glypicans, perlecan, neuroligins and the low-density lipoprotein receptors and their variants. AChE was observed to dock with growth arrest-specific protein 6 (Gas6) as well as apolipoprotein E3 (ApoE-3) at the same site as the laminin interaction. The AChE interaction site was shown to resemble the apolipoprotein-binding site on the low density lipoprotein receptor, and related molecules, including the low density lipoprotein receptor-related molecule (LRP) and the sortilin-related receptor (SORL1). These molecules, along with apoE, are associated with Alzheimer’s disease. Resemblances to the triggering receptor on myeloid cells (TREM1) were also suggested; this is interesting as AChE has been implicated in both haematopoiesis and haematopoietic cancers. Coimmunoprecipitation results, applied to investigate alternative ligands for AChE, confirmed the AChE-laminin interaction in neuroblastoma cells, and also suggested the existence of other binding partners. In conclusion, characterisation of the AChE-laminin interaction sites and investigation of structurally similar sites in other molecules suggests a role for AChE in the stabilization of the basement membrane of developing neural cells and provides a feasible explanation for the survival of the knockout mouse. Furthermore, the demonstrated similarity of the AChE interaction site to sites on molecules, notably the low density lipoprotein receptor family and SORL1 and their apolipoprotein ligands that are implicated in the pathology of Alzheimer’s disease, as well as the possible link to haematopoietic differentiation and cancers, warrants further investigation. / AFRIKAANSE OPSOMMING: Talle in vitro studies wys dat die ensiem asetielcholienesterase (AChE), behalwe vir sy klassieke rol in die hidrolise van asetielcholien (ACh), ‘n aantal nie-cholinerge rolle vertolk insluitend in sel adhesie, in die uitgroei van neurieten, in differensiering, asook in amyloidosis. Dit is vooraf gewys dat AChE, met behulp van elektrostatiese meganismes, in vitro met muis laminin-111 kan bind. Dit word verneem dat die area op AChE wat herken word deur monoklonale teenliggaampies (MAbs), moontlik ‘n kritiese area is met betrekking tot differensiasie. Dieselfde MAbs is gevind om beide die laminin-interaksie, sowel as sel adhesie van neuroblastoma selle, te inhibeer. In hierdie projek word die struktuur en eienskappe van die betrokke kritiese areas ondersoek deur die AChE-laminin interaksie te gebruik as sjabloon. ‘n Gedetailleerde analise van die teenliggaam epitoop het ook geskied. Met behulp van faag vertoon, modellering en hegting, sintetiese peptiede, ensiem-gekoppelde immunosorbent toetse (ELISAs) en konformasie interaksie area kartering, is die betrokke interaksie areas bestudeer. Hegting van enkel-ketting varierende fragment (scFv) volgordes, verkry vanaf die vaag vertoon, aan AChE dui dat die hoof herkennings motiewe die 90Arg-Glu-Leu-Ser-Glu-Asp motief, die 40Pro-Pro- Met-Gly volgorde, en die 59Val-Val-Asp-Ala-Thr-Thr (mens) motief is. ‘n Interaksie tussen muis AChE en die 1 G4 domein van laminin is gevind. Die interaksie betrek die basiese structure: Val2718-Arg-Lys-Arg-Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg- Lys2793; en Val2817-Glu-Arg-Lys2820. Die betrokkenheid van die AG-73 (2719-2729) area by hierdie interaksie is bevestig met ELISA eksperimente wat sintetiese peptiede inkorporeer. Die AG-73 area oorvleuel die heparin interaksie area op laminin. Hegtings eksperimente wys dat die hoof komponent van die interaksie area op AChE die suur volgorde Arg90-Glu-Leu-Ser-Glu-Asp95 op die omega-lus is. Die interaksie betrek ook die Pro40-Pro-Val42, Arg46 (gekoppel aan Glu94 deur ‘n sout-brug) en die heksapeptied Asp61 Ala-Thr-Thr-Phe-Gln66 motiewe. Analise van die MAb epitoop wys die hoof erkennings area as volgorde Pro40-Pro-Met-Gly-Pro-Arg-Arg-Phe48 (mens AChE). Die MAbs blyk ook gunstig te wees teenoor prolien-ryke volgordes soos Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 en Pro88-Asn-Arg-Glu-Leu-Ser-Glu-Asp95. Die areas betrokke by die AChElaminin interaksie is dus gedefinieer en ‘n moontlike rol vir hierdie interaksie in sel adhesie word voorgestel. Die noodsaaklikheid van AChE se nie-klassieke funksies word bevraagteken na die oorlewing van die AChE uitklop-muis. Resultate hier dui op die moontlikheid van funksionele oortolligheid as verduideliking hiervan, spesifiek met betrekking tot molekules betrokke in vroëe ontwikkeling asook in die proses van neurale agteruitgang. Deur gebruik te maak van die in vitro demonstrasies van die AChE-laminin interaksie, informasie verkry ten opsigte van die betrokke interaksie areas, asook resultate verkry vanaf die monoklonale teenliggaam (MAb) epitoop analise, word die idee van funksionele oortolligheid ondersoek. Hegtings en bioinformatika tegnieke is gebruik om molekules met soortgelyke strukture en uitdrukkings patrone in die embrioniese senuweestelses te ondersoek. Ko-immuno presipitasie tegnieke is gebruik om so moontlike alternatiewe ligande vir AChE te ondersoek. Moontlike funksionele oortolligheid van AChE met die volgende molekules is gevind: syndecan; glypican; perlecan; neuroligin; asook die lae-digtheid lipoproteien (LDL) reseptore en hul variante. Hegting van AChE met ’growth arrest-specific’ proteien 6 (Gas6) en die apolipoproteien E3 (apoE3) is gedemonstreer en gevind om dieselfde area as die laminin interaksie te betrek. Die betrokke interaksie area op AChE het ooreenstemminge met die apolipoproteien interaksie area op die LDL reseptor asook met verwante molekules soos die lae-digtheids lipoproteien reseptor-geassosieerde molekuul (LRP) en die sortilingeassosieerde reseptor (SORL1). Hierdie molekules, insluitend apoE, speel beduidende rolle in die patologie van Alzheimer se siekte. Ooreenkomste tussen AChE en die verwekkings reseptor op myeloïde selle (TREM1) is ook voorgestel, die interaksie is van belang siende dat AChE voorheen geassosieer is met beide haematopoiesis en haematopoietiese kankers. Ko-immuno presipitasie resultate bevestig die AChE-laminin interaksie en dui op die moontlike teenwoordigheid van alternatiewe ligande vir AChE in vivo. In konklusie, karakterisering van die AChE-laminin interaksie areas, gepaard met identifisering van struktureel ooreenstemmende areas in ander molekules, dui op ‘n rol vir AChE in die stabilisering van die basale membraan en verskaf dus ‘n geldige verduideliking vir die oorlewing van die AChE uitklop-muis. Die ooreenstemming van die AChE interaksie area met areas op ander molekules (spesifiek geassosieer met Alzheimer se siekte), asook die moontlike assosiasie van AChE met haematopoietiese differensiering en kanker, lê die grondslag vir verdere ondersoeke.

O câncer de mama é importante problema de saúde pública. O microambiente onde as células cancerígenas se encontram possui moléculas como a laminina e seus peptídeos bioativos, que influenciam a biologia tumoral. Estudo anterior realizado no laboratório demonstrou que o peptídeo C16, derivado da laminina, aumenta a expressão gênica de GPNMB e SPOCK1. Nesse trabalho, demonstramos que o peptídeo C16 aumentou níveis moleculares de GPNMB e SPOCK em células malignas MDA-MB-231e MCF-7, comparado com células normais MCF-10A. C16 estimulou significantemente a invasão de células MDA-MB-231. Silenciamento de GPNMB diminuiu a invasão celular desencadeada por C16. Contextualizando in vivo nossos resultados in vitro, imunohistoquímica em tissue microarrays mostrou que a presença de GPNMB e SPOCK é significantemente maior em câncer de mama. Assim, C16 regula os níveis de GPNMB e SPOCK em células mamárias malignas. C16 e GPNMB cooperam regulando a invasão de células MDA-MB-231. GPNMB e SPOCK foram mais detectados em câncer de mama comparado com mama normal. / Breast cancer is an important public health. The microenvironment in which cancer cells are found contains molecules such as laminin and its bioactive peptides that influence tumor biology. Previous study conducted in the laboratory showed that the C16 peptide derived from laminin, increases the gene expression of GPNMB and SPOCK1. In this work, we demonstrate that the C16 peptide increased molecular levels of GPNMB and SPOCK in malignant cells MDA-MB-231e MCF-7 cells compared with normal cells MCF-10A. C16 significantly stimulated invasion of MDA-MB-231 cells. GPNMB silencing decreased cell invasion triggered by C16. Contextualizing in vivo our in vitro results, tissue microarrays immunohistochemistry showed that the presence of GPNMB and SPOCK are significantly higher in breast cancer. Thus, C16 regulates the levels of GPNMB and SPOCK in malignant breast cells. C16 and cooperate GPNMB regulating the invasion of MDA-MB-231 cells. SPOCK and GPNMB were more detected in breast cancer compared to normal breast.

O câncer de próstata é o segundo câncer mais freqüentemente diagnosticado em homens. Durante o crescimento tumoral, as células neoplásicas interagem com a matriz extracelular (MEC). Analisamos o efeito de C16, peptídeo derivado da clivagem da MEC, sobre a migração, invasão e regulação dos invadopódios em células de câncer de próstata (DU145). Ensaios de migração e invasão demonstraram que C16 promoveu um aumento da atividade migratória e invasiva de células DU145 de maneira dose dependente. Demonstramos que o peptídeo estimula a fosforilação de Src. Ensaios de degradação em substrato fluorescente mostraram que C16 promoveu a formação de invadopódios de células DU145. O immunoblot nos revelou que este peptídeo também estimula a expressão de Tks4, Tks5, cortactina e MT1-MMP. C16 estimulou a produção de espécies reativas de oxigênio, importantes para o fenótipo invasivo das células tumorais. Nossos resultados sugerem que o peptídeo C16 regula migração, invasão, invadopódios e suas moléculas-chave e a geração de espécies reativas de oxigênio em células DU145. / Prostate cancer is the second most frequently diagnosed cancer in males. During tumor growth, neoplastic cells interact with the extracellular matrix (ECM) Our Laboratory has demonstrated that peptides derived from ECM cleavage are involved in migration, invasion and invadopodia formation in different tumor cell lines. Invadopodia activity depends on expression of the proteins Tks4, Tks5, cortactin, MT1-MMP, as well as reactive oxygen species (ROS) generation. Migration and invasion assays in chemotaxis chambers demonstrated that C16 increased migration and invasion activities of DU145 cells in a dose-dependent manner. We observed that the peptide stimulated phosphorylation of Src. Fluorescent substrate degradation assay showed that C16 increased invadopodia activity of DU145 cells. Immunoblot revealed that this peptide stimulated Tks4, Tks5, cortactin and MT1-MMP expression. Furthermore, C16 increased ROS production. Our results strongly suggested that C16 regulates migration, invasion, invadopodia key-molecules, and ROS generation in DU145 cells.

Au cours de la réparation cutanée, l’étape de réépithélialisation est essentielle car son objectif est de restaurer la fonction barrière de la peau. Elle consiste en une série d’étapes coordonnées où les kératinocytes migrent, prolifèrent et se différencient jusqu’à restauration complète de l’épiderme. Régulée de façon simultanée au niveau intracellulaire mais également extracellulaire, elle dépend de la production de facteurs de croissance, de métalloprotéases matricielles (MMPs) et de protéines de la matrice extracellulaire sur lesquelles les kératinocytes adhèrent et migrent par l’intermédiaire de récepteurs de la famille des intégrines ou des syndécans. Parmi les ligands matriciels, la laminine 332 (LN332), qui est connue comme étant la protéine d’adhésion majeure des kératinocytes de l’épiderme, s’avère être également impliquée au cours de la réépithélialisation et jouer un rôle important dans les processus d’adhésion et de migration des kératinocytes, notamment par le biais de son domaine globulaire LG4/5 localisé à l’extrémité C-term de sa chaine 3. De récentes études ont montré que ce domaine LG4/5 induit la migration des kératinocytes normaux humains (NHK), impliquant des MMPs pro-migratoires MMP-1 et MMP-9. Puisque les domaines LG4/5 ont été montrés comme participant à la dynamique du cytosquelette et au mouvement cellulaire par le biais des récepteurs syndécan-1 et -4, mon laboratoire d’accueil a décidé d’étudier l’implication du récepteur syndécan-1 dans ce processus d’expression de la MMP-9. Les analyses PCR et les résultats de zymographie obtenus ont révélé que le syndécan-1 joue un rôle dans l’expression et l’activation de la MMP-9 induite par le domaine LG4/5. De plus, la déplétion de l’expression du syndécan-1 dans les NHK a confirmé ces résultats. De précédents résultats de mon laboratoire d’accueil ont montré que le domaine LG4/5 induit la formation de filopodes médiée par le syndécan-1 au niveau du front de migration des kératinocytes. De ce fait, nous avons effectué des zymographies de gélatine in situ chez des NHK en migration afin d’observer la localisation de la MMP-9 et de savoir si cette dernière était trouvée au niveau de ces structures d’adhésion protrusives. Nous avons observé des zones de digestion de gélatine sous les NHK ressemblant à des points de contact d’adhésion. Leur nombre est augmenté chez des NHK traités avec le domaine LG4/5 de la LN332. L’utilisation d’inhibiteurs spécifiques des gélatinases et de la MMP-9 ont révélé que cette dernière est responsable de la formation de ces zones de digestion de gélatine. Parce que ces structures évoquent des podosomes, nous avons décidé de révéler leurs constituants majoritaires, à savoir la cortactine, la vinculine, l’-actinine, VASP, WASP ou encore Arp2/3. Dans le même temps nous avons également révélé le syndécan-1 afin de voir si ce dernier était présent dans ces structures. Nos résultats ont montré que tous les marqueurs des podosomes étaient localisés soit sous la forme d’un point à l’intérieur des zones de digestion pour les protéines régulatrices de l’actine, soit sous la forme d’un anneau entourant les zones de digestion pour les protéines d’adhésion et de signalisation associées à la membrane, confirmant donc que les structures observées sont bien des podosomes. Le syndécan-1 apparaît également sous une forme d’anneau, entourant les protéines régulatrices de l’actine et les zones de digestion laissant penser que le syndécan-1 serait impliqué dans ces structures. La diminution de l’expression du syndécan-1, en utilisant l’approche des petits ARN interférents dans des NHK, a montré que l’absence de syndécan-1 diminue de façon drastique le nombre et la surface des zones digérées. L’ensemble de nos résultats montre que le syndécan-1 serait un constituant des podosomes des kératinocytes, participant à leur formation et jouant un rôle dans le contrôle de l’expression de la MMP-9 / During skin repair, the reepithelialization step is essential to restore the skin barrier function. lt occurs by an orderly series of events whereby keratinocyte migrate, proliferate and differentiate until complete epidermal restoration. Keratinocyte migration determines the efficiency of the overall wound repair process. The keratinocyte's migratory behavior depends on the production of growth factors, matrix metalloproteinases (MMP) and on the dynamic interactions of the cells with extracellular components. Laminin 332 (LN332), known as a major adhesion substrate for keratinocytes, was shown to contribute to skin reepithelialization through its a3 chain C-terminal domains LG4/5. Recent studies have reported that LG4/5 induces keratinocyte migration, an event that relies on the involvement of the pro-migratory MMP-1 and MMP-9. As LG4/5 domains were shown to participate in cytoskeleton dynamic and cell movement through binding of the heparan sulfate proteoglycans syndecan-1 and -4, we analyzed the potential involvement of these receptors in this process. The PCR analysis and zymography results revealed that syndecan-1 plays a role in LG4/5 induced MMP-9 expression and activation. Down regulating or overexpressing syndecan-1 expression in cells confirmed these findings. As LG4/5 was shown to induce the formation of syndecan-1-mediated filopodia at the front of migrating cells, we performed in situ zymography experiments in migrating keratinocyte to analyze whether MMP-9 is found in these protrusive adhesion structures. Very interestingly, we found areas of digested gelatin resembling adhesion contacts underneath keratinocytes. Their number was increased in LG45-treated keratinocytes, a result in line with our previous data. The use of specific MMP inhibitors revealed that MMP-9 is responsible for the formation of these digested gelatin clusters. Further confocal microscope analysis revealed, at the cellular level, the presence of actin located within the digested areas, suggesting that an adhesion receptor would be involved in this process. Because these structures resemble podosomes, we revealed major podosome components, such as cortactin, vinculin, -actinin, VASP, WASP, Arp2/3 or dynamin and integrin. Our results showed all the podosome markers either within the digested areas (regulatory actin proteins) or organized as ring encircling the digested areas (signaling and adhesion proteins associated with plasma membrane), confirming that these structures are podosomes. Syndecan-1 also appears as a ring around the digested areas and encircling the regulatory actin proteins, suggesting that this receptor could be involved in these structures. The syndecan-1 depletion in normal human keratinocytes with specific siRNAs drastically decreased the digested areas surface. Taken together, our data demonstrate that syndecan-1 is a podosome components, participating in their formation and playing a role in MMP-9 expression and deposition. These results suggest that its re-distribution at the front edge of migrating keratinocyte may have a role to play in the cleavage or degradation of extracellular matrix proteins therefore facilitating their path through the fibrin clot

L’interaction du récepteur syndecan-1 de la famille des héparanes sulfates protéoglycanes avec le fragment carboxy-terminal alpha3LG4/5 de la protéine d’adhérence matricielle, la laminine 332, induit une réorganisation du cytosquelette de la cellule conduisant à la formation de filopodes et de microspicules, caractéristiques de la migration cellulaire. Notre laboratoire a mis en évidence que l’adhésion cellulaire syndecan-1 dépendante implique les chaînes d’héparanes sulfates et chondroïtine sulfate. Afin d’identifier le (les) zone(s) impliquée(s) dans l’interaction domaine LG4/5-syndécan-1, une approche de mutagenèse dirigée a été mise en place sur le fragment LG4/5 recombinant. Les résidus conservés parmi les laminines, identifiés dans la littérature comme liant l’héparine, aussi bien que des résidus basiques spécifiques à la chaine α3 identifiés par des approches prédictives, ont été remplacés par le résidu neutre glutamine. Toutes les protéines couplées avec l’étiquette 6-Histidine ont été produites dans des cellules de mammifère, purifiées par chromatographie d'affinité et caractérisées biochimiquement et par dichroïsme circulaire. L’évaluation de l’affinité des protéines produites pour l’héparine nous a permis d’identifier un site d’interaction majeur avec les glycosaminoglycanes dans le domaine LG4/5, entouré par des résidus à mineur affinité. La technique de résonance plasmonique de surface et des tests d’adhérence cellulaire nous ont permis de confirmer ce résultat puisque l’absence du site d’interaction majeur avec l’héparine a produit une inhibition totale de l’adhérence. Des expériences de pull-down nous ont montré que ce site est aussi impliqué dans l’interaction avec le syndecan-4, indiquant que cette séquence pourrait ainsi jouer un rôle dans différents processus cellulaires. Une collaboration avec des bio-informaticiens nous a permis de proposer un modèle structural du domaine LG4/5 et de montrer que la zone identifiée est localisée dans une boucle exposée à l’extérieure du module LG4, entourée par des résidus à plus faible affinité / The HSPG receptor syndecan-1 interacts with the carboxy-terminal LG4/5 domain in laminin 332 to participate in keratinocyte migration by inducing formation of cytoskeleton related protrusive structures. We have shown that syndecan-1 mediated cell adhesion occurs in heparan sulphate and chondroitin sulphate dependent manner and that these two glycosaminoglycan (GAG) chains bind independently to LG4/5 with different affinities. To identify residues involved in the interaction of the LG4/5 domain with syndecan-1 and to apprehend the molecular basis of the GAGs interaction specificity, we have used a site-directed mutagenesis approach of the recombinant LG4/5 fragment. The residues identified as conserved heparin binding residues throughout laminins, as well as “candidate” basic residues identified through predictive approaches, have been replaced by the neutral residue glutamine. All LG4/5 proteins carrying a hexa-histidine tag at their C-terminal end were expressed in mammalian cells. The produced proteins were purified and characterized biochemically. Circular dichroism studies performed on all mutagenised proteins showed that the overall structure of each mutant is comparable to that of the wild type protein. Heparin affinity chromatography analysis allowed us to identify a major heparin binding site in the LG4/5 domain surrounded by several minor GAG binding sites. Surface plasmon resonance analysis of mutated LG4/5 proteins-heparan sulphate interaction confirmed these results. These findings were well correlated with our in cellulo syndecan-1 mediated cell adhesion as the lack of this major heparin binding site totally abrogated cell adhesion. Pull down experiments allowed us to show that this heparin binding site sequence is responsible not only for the interaction of the receptors syndecan-1 but also for syndecan-4 suggesting that additional cellular functions may be carried by this sequence. Our structural predictions suggest that the LG4/5 in laminin 332 encompasses a major GAG binding site surrounded by a track of converging positively charged residues

O câncer de mama representa um importante problema de saúde pública. O microambiente onde as células neoplásicas se encontram desempenha importante papel na tumorigênese e progressão tumoral. Nosso Laboratório estuda o efeito da laminina, importante glicoproteína da matriz extracelular (MEC) e seus peptídeos dentro da biologia tumoral. Nesse projeto detectamos, in vitro e em amostras de câncer de mama humano, peptídeos derivados da clivagem proteolítica da laminina no câncer de mama. Inicialmente analisamos a distribuição das cadeias da laminina-111 no câncer de mama humano. Análise imuno histoquímica demonstrou aumento da marcação para as cadeias alfa1, beta1 e gama1 da laminina em tumores de mama. Células derivadas de câncer de mama (MDA-MB-231 e MCF-7) foram crescidas sobre Matrigel (substrato enriquecido em laminina). Células de mama normal MCF-10A serviram como grupo controle. Amostras controles e tratadas foram submetidas a eletroforese, immunoblot e espectrometria de massas (LC-MS/MS). Immunoblot mostrou a clivagem das cadeias da laminina in vitro e no câncer de mama humano, com a geração de fragmentos de menor massa molecular. Exploramos a liberação de peptídeos gerados pela clivagem da laminina em amostras de células tumorais em cultura e no câncer de mama humano. Análise proteômica mostrou que as bandas derivadas da laminina exibiram a presença de 31 peptídeos, entre eles C16 (KAFDITYVRL) e C28 (RVTLNRL). Previamente já demonstramos que o peptídeo C16 possui alta relevância na regulação tumoral. Diferentes peptídeos foram sintetizados e suas atividades biológicas avaliadas. Entre eles, C16 e C28 aumentaram significantemente a adesão, proliferação, secreção e ativação de MMPs além de invasão de células derivadas do câncer de mama (MDA-MB-231) e também de células derivadas de fibrossarcoma (HT1080). Investigamos também a ação do peptídeo C16 sobre as células tumorais MDA-MB-231 injetadas em zebrafish para avaliação do desenvolvimento tumoral in vivo. C16 promoveu aumento da área tumoral in vivo em comparação aos grupos controles. Concluímos que as cadeias alfa1, beta1 e gama1 da laminina estão aumentadas no câncer de mama. A clivagem da laminina possivelmente ocorre através da atividade de MMPs. As cadeias da laminina que são clivadas in vitro e no câncer de mama humano, geram fragmentos que contém peptídeos com funções biológicas evidentes. Entre os peptídeos observados, detectamos o peptídeo C16 in vitro e in vivo<i/>, também detectamos o peptídeo C28 in vitro. O peptídeo C16 é um grande regulador da invasão de células MDA-MB-231 e células HT1080, além de promover o desenvolvimento tumoral in vivo. Já o peptídeo C28 promove a proliferação de células MDA-MB-231 e HT1080. / Breast cancer represents an important public health problem. The microenvironment plays an important role in tumorigenesis and tumor progression. Our laboratory studies the effect of laminin, an important extracellular matrix glycoprotein (ECM) and its peptides in tumor biology. In this project we detected, in vitro and in human breast cancer samples, peptides derived from proteolytic cleavage of laminin. We initially analyzed the distribution of laminin-111 chains in human breast cancer. Immunohistochemistry demonstrated increased laminin alpha1, beta1 and gamma1 chains in breast tumors. Cells derived from breast cancer (MDA-MB-231 and MCF-7) were grown on Matrigel (laminin-enriched substrate). Normal breast cells MCF-10A served as a control group. Control and treated samples were subjected to electrophoresis, immunoblot and mass spectrometry (LC-MS/MS). Immunoblot showed cleavage of the laminin chains in vitro and in human breast cancer, with generation of lower molecular mass fragments. We explored the release of peptides, generated by laminin cleavage in tumor cell samples in culture and in human breast cancer. Proteomic analysis showed that the bands derived from laminin exhibited the presence of 31 peptides, among them C16 (KAFDITYVRL) and C28 (RVTLNRL). We have previously demonstrated that the C16 peptide has high relevance in tumor regulation. Different peptides were synthesized, and their biological activities were analyzed. Among them, C16 and C28 peptides, significantly increased adhesion, proliferation, secretion and activation of MMPs and cell invasion in breast cancer cells (MDA-MB-231) as well as fibrosarcoma-derived cells (HT1080). In addition, we investigated the action of the C16 peptide on zebrafish-injected MDA-MB-231 tumor cells for evaluation of tumor development in vivo. C16 promoted increase in tumor area in vivo compared to control groups. We conclude that, laminin alpha1, beta1 and gamma1 chains are increased in breast cancer. Laminin chains that are cleaved in vitro and in human breast cancer, generate fragments containing peptides with obvious biological functions. Among the peptides observed, we detected the C16 peptide in vitro and in vivo, we also detected the C28 peptide in vitro. C16 peptide is a major regulator of the invasion of MDA-MB-231 cells and HT1080 cells, in addition C16 promotes tumor development in vivo. C28 peptide promotes the proliferation of MDA-MB-231 and HT1080 cells.

<p>Migration of T-lymphocytes on a surface coated with extracellular matrix (ECM) components (two-dimensional (2-D) migration) and migration (infiltration) into a matrix (Three-dimesional (3-D) migration) are complex events and the underlying mechanisms are not yet fully understood. Here 2-D and 3-D migration were studied by use of seven leukemic T-cell lines representing discrete differentiation stages, a non-leukemic T-cell clone, and normal peripheral blood T cells. peripheral blood lymphocytes and the T-cell clone produced nanogram quantities of various chemokines, as compared to a production of ≤ 0.05 ng/ml by the T leukemia cell lines. In a Boyden chamber system, the leukemic T-cell lines showed haptotactic migration on fibronectin. The migration was augmented bu exposure to chemokines, including RANTES, MIP-1α, MIP-1β, and IL-8. The T-cell lines showed a peak response at a chemokine concentration of 10-50 ng/ml, whereas the T-cell clone responded optimally at 100 ng/ml. In contrast to a general capability of T-cells to migrate on 2-D ECM, only some of the T-cell lines were capable of 3-D migration into Matrigel or a collagen matrix. The infiltrative capacity was unrelated to the capacity to migrate on or adhere to the substrata. T-cell lines with a capacity to infiltrate produced matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), whereas non-infiltrating cell lines did not produce MMP-9. T-cell lines capable of infiltrating Matrigel or collagen responded to chemokines exposure with increased infiltration, but the chemokines did not render non-infiltrative cell lines infiltrative. Stimulation of infiltration of T-cell lines into collagen by the chemokine SDF-1α was inhibited by somatostatin, a neuropeptide with immunosuppressive properties. In conclusion, the ability to migrate on 2-D substrata and to infiltrate into 3.D substrata was found to be distinguishable properties of T cells. failure of some T-cell lines to infiltrate correlated with the lack of expression of MMP-9. Chemokines stimulated infiltration of infiltrative T-cell lines into collagen and Matrigel but did not render non-infiltrative T-cell lines infiltrative. Finally, a possible physiological mechanism for modulation of the chemokine-stimulated 3-D migration was demonstrated.</p>

<p>Basement membranes are thin extracellular sheets that surround muscle, fat and peripheral nerve cells and underlay epithelial and endothelial cells. Laminins are one of the main protein families of these matrices. Integrins and dystroglycan are receptors for laminins, connecting cells to basement membranes. Each laminin consists of three different chains, (α, β, γ). Laminin-1 (α1β1γ1) was the first laminin to be found and is the most frequently studied. Despite this, it was unclear where its α1 chain was expressed. A restricted distribution of the α1 chain in the adult epithelial basement membranes was demonstrated in the present study. In contrast, dystroglycan was found to have a much broader distribution. Dystroglycan is an important receptor for α2-laminins in muscle, but binds also α1-laminins. The more ubiquitous α5-laminins were also shown to bind dystroglycan, but with distinctly lower affinity than α1- and α2- laminins. </p><p>The biological roles of different laminin isoforms have been investigated. Differences were found in the capacity of various tested laminins to promote epithelial cell adhesion. The α5-laminins were potent adhesive substrates, a property shown to be dependent on α3 and α6 integrins. Each receptor alone could promote efficient epithelial cell adhesion to α5-laminins. Yet, only α6 integrin and in particular the α6A cytoplasmic splice variant could be linked to laminin-mediated activation of a mitogen-activated protein kinase (MAP kinase) pathway. Attachment to either α1- or α5-laminins activated extracellular-signal regulated kinase (ERK) in cells expressing the integrin α6A variant, but not in cells expressing α6B. A new role for dystroglycan as a suppressor of this activation was demonstrated. Dystroglycan antibodies, or recombinant fragments with high affinity for dystroglycan, decreased ERK activation induced by integrin α6 antibodies. Integrin αvβ3 was identified as a novel co-receptor for α5-laminin trimers. Cell attachment to α5-laminins was found to facilitate growth factor induced cell proliferation. This proliferation could be blocked by antibodies against integrin αvβ3 or by an inhibitor of the MEK/ERK pathway. Therefore, integrin αvβ3 binding to α5-laminins could be of biological significance.</p>

<p>The formation and maintenance of tissues in multicellular animals are crucially dependent on cellular interactions with the extracellular matrix (ECM). Two different studies on such interactions are presented herein.</p><p>Studies on expression of laminins in normal and dystrophic skeletal muscle, clarified a much debated issue regarding discrepancies seen for laminin α1-chain expression between human and mouse tissues. Lack of laminin α1-chain expression was verified in both mouse and human skeletal muscle. Furthermore, the earlier discrepancies seen for laminin α1-chain expression was explained by showing that an antibody-reagent, commonly used in human studies, recognised the laminin α5-chain rather than the laminin α1-chain</p><p>The integrin α11-chain (forming α11β1 integrin) is the latest addition to the integrin receptor family, and belongs to the I domain-containing group of integrin α-chains. Previous studies had shown that α11β1 is a collagen receptor. In the present study, the <i>in vitro</i> and <i>in vivo</i> functions of the α11-chain were further characterised. Distribution studies on embryonic human and mouse tissues showed that the α11-chain was expressed on mesenchymal cells in the developing tendon, perichondrium, intervertebral disc, and cornea. The interactions of α11β1 integrin with collagen type I and IV were studied <i>in vitro</i>. The α11β1 bound to these collagens in a manner similar to integrin α2β1 (with collagen type I being the preferred ligand for α11β1). Furthermore, α11β1 was shown to mediate migration on collagen type I coated surfaces, and to mediate contraction of collagen type I gels. The <i>in vivo</i> functions of the α11-chain were investigated by the generation of integrin α11-chain null-mice, using gene targeted disruption of the itga11 in embryonic stem cells. Two independent lines of mice lacking α11 protein were generated. Phenotypic analysis of these mice indicated a role for α11β1 in the formation of the musculoskeletal system.</p>

<p>Blood vessels transport blood with essential nutrients and oxygen to the cells in our body. In a healthy adult, formation of new vessels (angiogenesis) occurs only in case of tissue repair and growth. Physiological angiogenesis requires precise regulation of multiple signaling components, a process which is deregulated in a number of pathological conditions, such as cancer. This thesis is focused on the role of laminins, heparan sulfate proteoglycans (HSPGs) and vascular endothelial growth factor (VEGF)-A in regulation of vascular development and angiogenesis. As a model, we have used embryonic stem cells that differentiate to form blood vessels in a manner faithfully recapitulating the <i>in vivo</i> processes. </p><p>We show that the basement membrane (BM) protein laminin-111 promotes maturation of endothelial cells in the presence of fibroblast growth factor-2, a known endothelial cell mitogen. However, embryonic stem cells are able to differentiate into endothelial cells also in the absence of laminin deposition in the vascular BM. Sprouting angiogenesis, induced by VEGF-A, is also not strictly dependent on laminin deposition. On the other hand, in the absence of laminins, vessels are enlarged. These data suggest an important role for laminins in regulation of the vessel diameter.</p><p>We also show that HSPGs serve as coreceptors for VEGF-A to regulate vascular development. The mode of presentation of HSPGs, <i>in</i> <i>cis</i> (on the endothelial cell) or <i>in</i> <i>trans</i> (on an adjacent cell such as pericytes), is critical in regulation of VEGF receptor-2 activation and stimulation of vascular development. Binding of VEGF-A to HSPGs <i>in</i> <i>trans</i> leads to accumulation of activated VEGFR-2 in endothelial cells and to prolonged signaling. This demonstrates a potential role for HSPGs in regulation of receptor trafficking and signaling kinetics, with possible implications also for other HS-binding ligand/receptor systems.</p>