Dissertations / Theses on the topic 'Extracellular proteolysi'

Urokinase (uPA) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homeostasis. Moreover the uPA-system plays an important role in many pathological events, such as tumour cell migration and dissemination. On the one hand, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. On the other hand, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin (VN). Although the existence of feedback loops between the functions of uPAR in extracellular proteolysis, cell adhesion and signalling has been described, some aspects of this cross-talk are still poorly understood and not characterized experimentally. We here report that cell surface plasminogen activation induces a potent negative feedback on cell adhesion to VN. The feedback is predominantly caused by proteolytic cleavage of the RGD-motif in VN catalyzed by both uPA and plasmin. In this process the cell-adhesive properties of VN are impaired by disruption of the integrin binding site and release of the somatomedin B (SMB) domain responsible for binding of uPAR. Cleavage of VN by uPA displays a remarkable receptor-dependence and requires concomitant binding of both uPA and VN to uPAR suggesting that the hydrolysis is accelerated by a mechanism of substrate presentation. VN represents the first described uPAR-dependent substrate of uPA and our findings therefore identify a potential novel function of uPAR in focusing the proteolytic activity of the plasminogen activation system onto extracellular matrix-associated VN. Additionally, SMB-containing N-terminal VN fragments are released by several cancer cell lines in vitro and are detectable in human urines samples. We have thus developed a clinical grade immunoassay for the detection and quantification of such fragments in urine samples with the aim of using the levels of urinary VN fragments as a novel cancer biomarker. Our working hypothesis is that this biomarker may be used as an indirect functional measurement of the uPA-system activity in the tumour tissue. Finally, we show that the specific urokinase inhibitor, plasminogen activator inhibitor 1 (PAI-1), blocks the negative feedback mediated by uPA and behaves as a potent uPA-dependent agonist of the interaction between uPAR and VN. Indeed, we report for the first time that the covalent complex between uPA and PAI-1 is endowed with higher agonistic activity compared to uPA. Taken together, these data might represent a molecular explanation of the poor clinical outcome observed in cancer patients with high levels of uPA and PAI-1.

Le muscle squelettique est le réservoir principal d’acides aminés libres de l’organisme. Ainsi, l’atrophie musculaire induite par l’immobilisation peut entraîner un affaiblissement et un allongement des périodes de récupération générant des coûts de santé publique élevés. Une aggravation de l’atrophie caractérise de façon surprenante le muscle tibialis anterior (TA) après le déplâtrage, retardant la récupération. Mon objectif a été de comprendre les mécanismes à l’origine de l’aggravation de l’atrophie du TA pendant les phases précoces de récupération en étudiant i) la structure et le phénotype des muscles, ii) la composition de la matrice extracellulaire (MEC), iii) la protéolyse et l’apoptose, et iv) les processus de signalisation via les intégrines. Des rats ont été soumis à une immobilisation par plâtrage pendant 8 jours d’une des deux pattes arrière, l’autre servant de témoin, et placés en récupération pendant 10 jours. L’aggravation de l’atrophie du TA apparaît dès déplâtrage, corrélée avec i) une baisse de l’aire des fibres associée à leur déformation, ii) une redistribution des isoformes des chaines lourdes de myosines, iii) une augmentation de l’apoptose localisée dans le tissu conjonctif, iv) un épaississement de l’endomysium pendant la remobilisation, v) des adaptations au niveau des processus de remodelage des collagènes, et vi) une activation prononcée et persistante du système protéolytique ubiquitine-protéasome (UPS) et de l’apoptosome. Nous montrons également une élévation des niveaux ARNm dans le TA remobilisé vii) de la ténascine-C et de Sparc dès le déplâtrage, et viii) de marqueurs de l’autophagie à partir du moment où l’atrophie se stabilise. Enfin, nous montrons également une élévation des ARNm dans le TA immobilisé ix) des facteurs myogéniques, et x) des intégrines membranaires et de leurs partenaires pendant l’immobilisation et après le déplâtrage. En conclusion, mon travail de thèse a permis de montrer que l'aggravation de l’atrophie du TA est précoce, associée à un remodelage important de la structure et de la composition de la MEC et du phénotype des fibres musculaires, et pourrait résulter de l’augmentation persistante et prononcée de la voie UPS et de l’apoptose. Ce travail suggère que des modifications au niveau des molécules matricielles pendant la remobilisation pourraient influencer la signalisation dépendante des intégrines et la régénération musculaire
Skeletal muscle is the main reservoir of body amino acids. Thus, muscle atrophy induced by immobilization can lead to a weakening and to a lengthening of recovery periods, leading to elevated healthcare costs. Surprisingly, a worsening of tibialis anterior (TA) muscle atrophy prevailed after cast removal and thus delayed recovery. The aim of my Ph.D was to understand mechanisms underlying the worsening of TA atrophy during early recovery by studying i) the muscle structure and phenotype, ii) the composition of the extracellular matrix (ECM), iii) proteolysis and apoptosis, and iv) the signaling pathways via integrins. Rats were subjected to hindlimb casting for 8 days of one hindllimb, the other leg served as control, and then were allowed to recover for 10 days. The worsening of TA atrophy appeared immediately after cast removal and correlated with i) a decrease in fiber crosssection area associated to fiber deformation, ii) a redistribution of myosin heavy chain isoforms, iii) an increase in apoptosis localized in the connective tissue, iv) a thickening of the endomysium during remobilization, v) some adaptations in collagen remodeling processes, and vi) a pronounced and sustained activation of the ubiquitin-proteasome proteolytic system (UPS) and of the apoptosome. We also showed an increase in the remobilized TA of mRNA levels vii) of tenascin-C and Sparc immediately after cast removal, and viii) of some autophagy markers, when atrophy stabilized. Finally, we showed an elevation of mRNA levels encoding ix) myogenic factors, and x) transmembrane integrins and their partners during TA immobilization and after cast removal. In conclusion, my Ph.D project showed that the worsening of the TA atrophy occurred early after cast removal, was associated with a significant remodeling of the structure and composition of the ECM and of the phenotype of muscle fibers, and may result from pronounced and sustained increase in the UPS and apoptosis. This work suggests that changes in the matricellular matrix molecules during remobilization could influence integrin-dependent signaling and muscle regeneration

Neutral proteinases, released by inflammatory leukocytes, have been implicated in the pathogenesis of pneumoconiosis but there has been no systematic study of the proteolytic acitivty of leukocytes from dust-exposed lung. The aim of the present study was, therefore, to assess the bronchoalveolar leukocyte profile and proteolytic activity of the leukocytes in a rat model of pneumoconiosis. An assay, based on the breakdown of [¹²⁵I] fibronectin, that would measure the overall proteolytic activity of the bronchoalveolar leukocytes and indicate their potential to damage the connective tissue of the alveolar septum was developed and validated. Increased proteolytic activity was found in the inflammatory bronchoalveolar leukocyte populations and so the relative role of macrophages and neutrophils was assessed by separation into distinct populations; both inflammatory macrophages and neutrophils had increased proteolytic activity. The important features governing the inflammogenicity of particles were addressed by measuring the inflammation-generating properties of a variety of fibrogenic and non-fibrogenic particles in rats exposed by intratracheal instillation or inhalation. The number of leukocytes recruited to the alveolar region was measured by bronchoalveolar lavage. Only fibrogenic mineral dusts had the ability to produce a sustained alveolitis in which the proteolytic activity of the bronchoalveolar leukocytes remained elevated. The pathogenicity of fibrogenic particles is likely, therefore, to be related to their ability to evoke and sustain an increased lung proteinase burden. The sustained alveolitis with fibrogenic particles was not related to lack of clearance of dust from the lung. Both quartz and coalmine dust elicited persistent alveolitis, but titanium dioxide, which is no more readily cleared than silica or coalmine dust, failed to sustain the inflammation. Properties of the particle surface, at least in the case of quartz, appear to play a part in their inflammogenicity. Altering the surface of quartz particles by coating them with aluminium lactate reduced their ability to recruit inflammatory leukocytes but did not alter the proteolytic activity of the leukocytes. The tissue response to the aluminium-coated quartz particles was also less than that elicited by native quartz with fewer and less-severe lesions. The foregoing serve to substantiate the role of inflammatory leukocytes in the pathogenesis of pneumoconiosis. Only when there is a sustained alveolitis with an overall increased proteinase burden, does pathological change occur in the lung. Reducing the magnitude and/or the duration of the alveolitis markedly suppresses the development of the tissue lesions.

Bibliography: pages 161-176.
Vibrio alginolyticus is a gram-negative aerobic bacterium that produces several extracellular serine proteases and a collagenase during the stationary growth phase. The aim of this study was to investigate alkaline serine protease production by this organism, and to attempt the cloning and expression of a V.alginolyticus protease gene in Escherichia coli.

Nous étudions la migration et la protéolyse de la matrice extracellulaire dans le cancer du sein. Pour cela, nous avons mis en place deux systèmes modèles. Le premier, se base sur une lame basale reconstituée et permet d'évaluer le potentiel invasif de lignées cellulaires tumorales. Nous montrons que les cellules cancéreuses migrent différemment à travers un gel pour former des amas de taille variable directement corrélé à leur pouvoir invasif. Dans notre système, seule la migration de type mésenchymateuse est utilisée par les cellules. Ce type de mouvement est directement dépendant de protéases sécrétées par les cellules. Nous avons, donc mesurée la synthèse au niveau transcriptionnel de la classe d'enzyme majoritairement impliquée dans la dissémination tumorale, les matrice métalloprotéases (MMPs). Nous avons ainsi pu montrer que l'expression de 3 MMPs est corrélée aux capacités migratoires des cellules donc à leur potentiel invasif. Le processus physique par lequel les enzymes dégradent les matrices est très peu étudié au niveau expérimental. Le second système que nous utilisons se base sur un modèle de matrice conjonctive majoritairement composer de collagène de type I. Nous utilisons la gélatine, pour étudier la protéolyse de gels protéiques par différentes classes de protéases. A partirdes études sur la solubilisation enzymatique des gels par l'-chymotrypsine, la protéinase K et la papaïne, nous montrons qu'il existe des mécanismes de dégradation distincts. Le premier est un mécanisme anormal dont la cinétique est limitée par la diffusion de l'enzyme, le second est brownien et la cinétique est limitée par la réaction. Ce second mécanisme dépend directement d'interactions eléctrostatiques entre l'enzyme et le gel. Nous observons pour deux des enzymes que l'évolution des temps de dégradation mais également la cinétique dépendent de la concentration en protéine dans les gels
We study the migration and proteolysis of the extracellular matrix in breast cancer. For this, we set up two model systems. The first is based on a reconstituted basement membrane and allows the evaluation of invasive potential tumor cell lines. We show that cancer cells migrate differently across the gel to form clusters of variable size directly correlates with their invasiveness. In our system, only the migration of mesenchymal type is used by the cells. This type of movement is directly dependent proteases secreted by the cells. We therefore measured the synthesis at the transcriptional level of the enzyme class mainly involved in tumor dissemination, the matrix metalloproteases (MMPs). We were able to show that the expression of 3 MMPs is correlated with migratory capacity of cells, therefore their invasive potential. The physical process by which enzymes degrade the matrix is very little studied at the experimental level. The second system we use is based on a model of connective matrix mainly composed of collagen type I. We use gelatin for the study of protein gels proteolysis by different classes of proteases. Based on the study of gels enzymatic solubilization by a- chymotrypsin, proteinase K and papain, we show that there are distinct mechanisms of degradation. The first mechanism is abnormal whose kinetic is limited by enzyme diffusion, and the second is Brownian and the kinetic is reaction limited. The second mechanism depends directly on electrostatic interactions between enzyme and gel. We observe for two enzymes that the evolution of degradation time but also the degradation kinetics depend on the concentration of protein in gels

In condizioni patologiche le gelatinasi o meglio la MMP-2 e la MMP-9 vengono espresse dalle cellule in quantità più elevate rispetto alle normali condizioni fisiologiche. Ne sono un esempio i processi infiammatori e la progressione tumorale, dove queste endopeptidasi Ca+2 e Zn+2 dipendenti sono attivamente coinvolte nel processamento delle membrane basali cellulari; infatti la loro azione permette alle cellule tumorali la loro progressione all’interno di nuovi tessuti, dando origine alla formazione di metastasi. Le gelatinasi influenzano la progressione tumorale in quanto intervengono in un processo che prende il nome di “angiogenic switch”, che consiste nella formazione di una nuova vascolarizzazione in grado di alimentare le cellule in attiva proliferazione. L’ ”angiogenic switch” inizia con la degradazione da parte delle gelatinasi del collagene IV, componente fondamentale della membrana basale vascolare. Esso, una volta processato, libera fattori (quali il VEGF) che stimolano le cellule endoteliali a migrare all’interno di una matrice provvisoria dove si formeranno i nuovi vasi sanguigni. Se da un lato le gelatinasi aumentano l’attività proliferativa delle cellule tumorali, dall’altra sono in grado di inibirla. Sembra infatti che la MMP-2, degradando il collagene di tipo IV, produca dei frammenti con attività anti-angiogenica in grado di bloccare la proliferazione tumorale. Ai fini di una miglior comprensione del ruolo delle Gelatinasi durante la progressione tumorale, abbiamo analizzato l’efficienza delle due metalloproteinasi verso il collagene di tipo IV; e nel caso della MMP-2 abbiamo caratterizzato il ruolo dei suoi diversi domini verso questo substrato naturale. Inoltre è stata effettuata una analisi basata sulla capacità dei neutrofili di migrare attraverso una membrana ricoperta di collagene di tipo IV in presenza di (i) una quantità esogena crescente di MMP-2 (ii) frammenti di arrestina derivanti dal processamento della catene  del collagene IV, (iii) MMP-2 mutata, ossia priva del suo dominio catalitico, (iv) del dominio ricombinante fibronectinico della MMP-2 o rCBD e (v) la MMP-2 inattiva o pro-MMP-2.Per finire abbiamo studiato l’esistenza di una possibile interazione tra le due gelatinasi nel processare il collagene di tipo IV sia in vitro che in condizioni ex vivo.I nostri risultati dimostrano come entrambe le gelatinasi processano il componente fondamentale delle membrane basale vascolare in condizioni fisiologiche, 37°C e pH 7,2. Inoltre l’MMP-2 interagisce con la MMP-9 aumentandone l’efficienza di processamento verso il collagene di tipo IV, per effetto di un controllo allosterico dovuto al semplice legame dell’enzima con il substrato. Quando la MMP-2 si lega al collagene IV per opera del suo dominio fibronectinico (CBD) , la MMP-9 processa il substrato con maggiore efficienza rispetto all’ azione quando è prsente da sola. Il fenomeno varia a seconda della concentrazione della MMP-2 che possiede due siti di legame per questo tipo di collagene: uno ad alta affinità ed un secondo sito con una scarsa affinità di legame. A bassa concentrazione di MMP-2 viene legato solo il sito del substrato per cui l’enzima posside una maggior affinità e di conseguenza la MMP-9 legandosi allo stesso substrato lo processa con maggior efficienza. Al contrario, ad alte concentrazioni di MMP-2, l’enzima si lega ad ambedue i siti di attacco che possiede nel collagene IV diminuendo la capacità della MMP-9 di processarlo, a causa di una probabile sovrapposizione del sito di attacco dei due enzimi.L’attività sinergica delle due gelatinasi viene riscontrata anche ex vivo, ossia mediante esperimenti di migrazione cellulare attraverso un coating di collagene IV. Le cellule utilizzate sono neutrofili, cellule in grado di esprimere vari enzimi proteolitici tra cui metalloproteinasi come la MMP-8 e la MMP-9 e proteasi a serina come l’elastasi del neutrofili. Risultano invece non produrre livelli apprezzabili di MMP-2, come dimostrato dalle zimografie delle Figure 10 e 11. I neutrofili, grazie alla MMP-9, sono quindi in grado di oltrepassare la barriera di collagene IV, che viene processato con maggior attività in presenza di quantità esogene di MMP-2 priva del dominio catalitico o in aggiunta del suo dominio di legame al substrato, il dominio ricombinante rCBD.
Proteolytic degradation of basement memnbrane influences the cell behaviour during important processes, such as inflammations, tumorigenesis, angiogenesis and allergic diseases. In this study, we have investigated the action of gelatinase A (MMP-2) and B (MMP-9) on collagen IV, the major constituent of the basement membrane. We have compared quantitatively their actions on the soluble forms of collagen IV extracted with or without pepsin (from human placenta and from Engelbreth-Holm-Swarm (EHS) murine sarcoma, respectively). The catalytic efficiency of MMP-2, and also MMP-9, is dramatically reduced in the case of the EHS murine sarcoma with respect to the human placenta, probably due to the much tighter packing of the network which renders very slow the speed of the rate-limiting step. We have also enquired on the role of MMP-2 domains in processing collagen IV. The removal of the hemopexin-like domain, using only the catalytic domain of MMP-2, has only a limited effect on the catalytic efficiency toward collagen IV, indicating that the missing domain has not a great relevance for the overall mechanism. Instead, the addition of the isolated collagen binding domain, corresponding to the fibronectin-like domain of whole MMP-2, greatly inhibits the cleavage process, demonstrating that MMP-2 interacts with collagen type IV preferentially through its fibronectin-like domain. Finally, we have investigated the effect of MMP-2 proteolytic activity ex vivo. MMP-2 action negatively affects the neutrophil cells migration across type IV coated membranes and this is likely related to the production of lower molecular weight fragments which impair the cellular migration.However, for both types of collagen IV the enzymatic processing by MMP-9 is dramatically enhanced in the presence of the Collagen Binding Domain of Gelatinase A (CBD). This effect, clearly indicates that the fibronectin-like domain of MMP-2 and MMP-9 bind to topologically distinct sites on type IV collagen, bringing about a conformational change of the collagen IV molecule. This allows the two enzymes to cooperate with each other through a ligand-linked mechanism, which does not necessarily require the enzymatic action. Therefore, fibronectin-like domains not only increase the affinity between enzyme and substrate to enhance the catalysis, they also act as allosteric third party elements in the MMP action. This synergistic action between MMP-2 and MMP-9 on collagen IV has been tested also with an ex vivo eperiment. The MMP-2 without the catalytic domain, the rCBD and the pro-MMP-2 increase the neutrophil cells migration across collagen IV coated membranesand this is related to the growth of the catalytic activity of MMP-9.

Extracellular matrix (ECM) modifications occur during plant growth, development, and in response to environmental stimuli. Key modulators of ECM modification in vertebrates, the extracellular matrix metalloproteinases (MMPs), have also been described in a few plants. Here, we report the identification of Loblolly pine (Pinus taeda) Pta1-MMP and its characterization during seed development and germination. The Pta1-MMP protein has the structural characteristics of other plant MMPs, and a recombinant protein (rPta-MMP) generated by using EST sequences for a seed-expressed MMP exhibits Zn2+-dependent protease activity, and is inhibited by the active site-binding hydroxamate inhibitor GM6001 and EDTA. The Pta1-MMP gene is expressed during embryo development, with transcript levels increasing from proembryo to early cotyledonary stage, then declining during late cotyledonary expansion and maturation drying. Protein extracts exhibited similar developmental-stage MMP-like activity. Seed imbibition in water facilited germination, which was stimulated by GA3 and inhibited by ABA. The timing of germination was mirrored by the presence of MMP-like protease activity in both water- and GA3-imbibed embryos. Pta1-MMP transcript levels increased in association with germination for both GA3- and water-treated embryos, in agreement with MMP-like activity. In contrast, by 10 days after imbibition, Pta1-MMP transcripts in ABA-treated embryos were at levels similar to the other treatments, although MMP-like activity was not observed. The application of GM6001 during Loblolly pine seed imbibition inhibited germination in a dose-dependent manner. Our results suggest that Pta1-MMP is required for ECM modification, facilitating the cell division and expansion required for both embryo development and germination. To our knowledge, this is the first report of an MMP in any gymnosperm and also its involvement in embryo development and subsequent germination.
Ph. D.

Les metalloproteinases matricielles (mmps) sont impliquees dans tous les processus de proteolyse matricielle, qu'ils soient physiologiques ou pathologiques. Au cours du developpement tumoral dans le cancer de vessie, le passage au travers de la membrane basale depend de deux mmps : les gelatinases a et b (mmp-2 et mmp-9). Une methode de purification par electrophorese preparative a permis de caracteriser les differentes formes de gelatinases urinaires et de montrer que les taux de gelatinase augmentent significativement dans le cancer de vessie alors que les inhibiteurs tissulaires associes (timp-1 et timp-2) diminuent ; les rapports mmps/timps, reflet de l'equilibre proteases/inhibiteurs, sont proposes comme marqueurs de diagnostic et de pronostic (seuil 0,9%). Parallelement a cette etude, la ngal (neutrophil gelatinase associated lipocalin), une lipocaline qui forme avec la mmp-9 un complexe dimerique, a ete mise en evidence dans les urines. Nous avons montre que l'excretion de ngal dans les urines est un marqueur d'evolution du cancer de vessie : son taux diminue significativement pour les tumeurs de mauvais pronostic. Une approche plus fondamentale de l'etude du timp-1 a permis d'aborder ses differents roles dans des modeles cellulaires (rt 112 et t 24) de cancer de vessie. Nous avons montre, dans des experiences in vitro de migration cellulaire a travers une couche de matrigel que le timp-1 est un inhibiteur de l'invasion tumorale. Ainsi, le timp-1 penetre dans ces cellules : il pourrait exercer une regulation de type autocrine ou paracrine. La proteine hybride egfp-timp-1 fluorescente a ete visualisee par microscopie au cours de sa penetration et de son adressage nucleaire. Ces experiences de ciblage nucleaire du timp-1 suggerent un role pour le timp-1 dans la regulation des cellules tumorales, role qui reste a elucider.

Xylella fastidiosa é uma bactéria patogênica encontrada em várias plantas. Esta bactéria secreta proteases extracelulares detectadas em gel de eletroforese, sendo a gelatina usada como substrato co-polimerizado. Três principais bandas protéicas foram detectadas com massa molar (MM) de 122, 84 e 65 kDa produzidas pelo isolado de citros (X0) e duas bandas de aproximadamente 84 e 65 kDa de isolado de videira (9713). Estas bactérias produziram zonas de hidrólise em meio sólido contendo gelatina, caseína e hemoglobina. Os resultados usando a gelatina como substrato foram os melhores para a atividade das proteases. A atividade enzimática das proteases de X. fastidiosa de citros e videira foi completamente inibida por PMSF e parcialmente inibida por EDTA, podendo ser visualizado em gel de eletroforese nativo. A temperatura ótima de atividade protéica foi de 30oC e o pH ótimo de 7,0. Além das proteases secretadas por este fitopatógeno, quitinase e β-1,3-glucanase foram também detectadas no sobrenadante das culturas. Os resultados sugeriram que estas proteases produzidas pela X. fastidiosa de citros e videira pertencem ao grupo das serina e metalo proteases.
Xylella fastidiosa is a pathogenic bacterium found in several plants. These bacteria secrete extracellular proteases into the culture broth as visualized in sodium-dodecyl-sulfate polyacrylamide activity gels containing gelatin as a co-polymerized substrate. Three major protein bands were produced by strain X0 (citrus) with molar masses (MM) of 122, 84 and 65 kDa. Grape strain 9713 produced two bands of approximately 84 and 64 kDa. These organisms produced zones of hydrolysis in agar plates amended with gelatin, casein and hemoglobin. Gelatin was the best substrate for these proteases. SDS-PAGE activity gel indicated that the protease activities of X. fastidiosa from citrus and grape were completely inhibited by PMSF and partially inhibited by EDTA. The optimal temperature for protease activity was 30oC with an optimal pH of 7.0. Among the proteolytic enzymes secreted by the phytopathogen, chitinase and β-1,3-glucanase activities were also detected in cultures of X. fastidiosa (citrus). From these results, it is suggested that these proteases produced by strains of X. fastidiosa from citrus and grape, belong to the serine- and metallo-protease group.

Abstract Angiopoietins 1 and 2 (Ang1 and Ang2) are the ligands of the Angiopoietin/Tie signalling system, which is a binary pathway offering mechanisms for healthy vessels to reach and maintain their quiescence but also to rapidly respond to activating stimuli leading to a remodelling of endothelium. The latter is linked to disease settings such as inflammation and cancer where endothelial cell (EC) integrity is compromised and is often related to an increase in Ang2 expression. This study focused on the mechanisms enabling Ang1 to mediate both EC stability and migration and molecular and cellular determinants for ligand-specific functions of Ang2 and its isoform Ang2443. The findings revealed that Ang1 induces differential signalling depending on whether it anchors and activates Tie2 in cell-cell junctions in quiescent ECs, or in cell-matrix contacts in mobile ECs, thus leading to cellular phenotypes characteristic of resting and mobile ECs, respectively. In the second part of the thesis Ang2-Tie2 specific cell-extracellular matrix (ECM) contact sites were studied. Formation of Ang2/Tie2 EC-ECM contact sites was dependent on the collagen I and IV matrices, low Ang2 oligomerization state, α2β1-integrins, and intact microtubules. In the third part of the thesis the comparison of Ang2 mRNA splice variant Ang2443 with full length Ang2 (Ang2FL) revealed both redundant and ligand form–specific effects, expression of Ang2443443 increased the amount of monomeric ligand forms due to proteolytic processing and promoted transendothelial migration of cancer cells in vitro. On the other hand, both Ang2443 and Ang2FL were stored in endothelial Weibel-Palade bodies (WPBs), similarly induced Ang2-specific Tie2 cellular redistribution, and were mostly comparable in developmental angio- and lymphangiogenesis
Tiivistelmä Angiopoietiinit 1 ja 2 (Ang1 ja Ang2) ovat Ang/Tie signalointireitin kasvutekijöitä. Ang1 kasvutekijää tarvitaan sydämen ja verisuoniston sikiöaikaiseen kehittymiseen, se vähentää Tie2 reseptorin kautta verisuonten läpäisevyyttä, mutta edistää myös yksittäisten endoteelisolujen liikkumista. Saman Tie2 signalointireitin toisen kasvutekijän Ang2:n ilmeneminen johtaa verisuonten läpäisevyyden kasvuun tulehduksessa, uusien verisuonten muodostumiseen syöpäkasvaimissa ja syöpäsolujen leviämiseen elimistössä. Väitöskirjatutkimuksessa selvitettiin niitä solutason mekanismeja, joilla Ang1 kykenee välittämään sekä endoteelisolujen tiiviyttä että liikkumista. Lisäksi tutkittiin niitä molekyyli- ja solutason mekanismeja, joilla Ang2 ja sen isomuoto Ang2443 välittävät kasvutekijäspesifisiä vaikutuksiaan. Väitöskirjassa osoitettiin että Tie2 reseptori paikantuu verisuonten endoteelisoluissa Ang1 sitoutumisen seurauksena joko solu-soluliitoksiin, tai yksittäisissä endoteelisoluissa solu-soluväliaine rajapinnalle. Tie2:n siirtyminen solu-soluliitoksiin aktivoi soluissa signalointireittejä, jotka ovat tyypillisiä normaaleille tiiviille verisuonille ja solu-soluväliaineliitoksissa liikkuville endoteelisoluille tyypillisiä piirteitä. Väitöskirjatyön toisessa osassa tutkittiin Ang2:lle ominaisia vaikutuksia ja Ang2-Tie2 kompleksin paikantumista erityisiin solu-soluväliaineliitoksiin. Tämä oli riippuvaista Ang2:n oligomerisaatiosta, kollageenisoluväliaineesta, α2β1-integriinistä ja normaalista mikrotubulusverkostosta. Väitöskirjatyön kolmannessa osassa osoitettiin että Ang2443 isomuodolla on sekä yhteisiä että isomuotospesifisiä piirteitä verrattuna kokopitkään Ang2:een (Ang2FL). Liukoinen Ang2443, mutta ei Ang2FL, esiintyi yleisesti monomeerisenä ligandimuotona proteiinin multimerisaatio-osan pilkkomisen seurauksena. Ang2443 lisäsi myös syöpäsolujen liikkumista endoteelisolujen läpi. Toisaalta sekä Ang2443 että Ang2FL varastoitiin endoteelisoluissa Weibel-Palade varastokappaleisin, ne välittivät samanlaista Tie2 reseptorin paikantumista endoteelisoluissa ja toimivat pääsääntöisesti samanlaisina kasvutekijöinä veri- ja imusuonten kehityksen aikana hiiressä

A variety of proteases have been shown to promote the progression of cancer by virtue of their ability to degrade extracellular proteinaceous barriers, such as basement membrane and interstitial stroma. At the outset of this study available evidence strongly implicated cathepsin D in breast cancer metastasis. It was envisaged that an antibody inhibitory to the activity of this enzyme might retard invasion, and restrain a tumour from spreading. To this end anti-peptide antibodies were generated against a peptide sequence derived from the substrate capturing "flap" of the enzyme. Inhibition of enzyme activity by these antibodies could not be demonstrated, probably due to the lack of a suitably sensitive enzyme assay. However, the rationale of this study and the expertise gained from it could be applied, in the future, to enzymes that have since been found to be more relevant to tumour invasion. A feature of many transformed cells is an anomalous lysosomal enzyme trafficking system, and concomitant hyper-secretion of some enzymes. The distribution of low pH compartments and lysosomal enzyme-containing compartments was investigated in human breast epithelial cells, and their c-Ha-ras- transformed counterparts. Immunofluorescence and immunoelectron microscopy showed that these compartments have a more peripheral cellular distribution with respect to normal cells, and cathepsins B and D were cell surface-associated. Studies were undertaken to reveal the extracellular matrix degrading ability of c-Ha- ras-transformed cells. Transformed cells exhibited increased degradation of fluorescein-labelled extracellular matrix in serum free medium, and increased motility, and degradation and disruption of extracellular matrix in serum-containing medium. In vitro invasion through artificial basement membrane by transformed cells was investigated using scanning electron microscopy, and was further used to preliminarily identify the proteases involved in invasion by specific inhibition. By this means, greatest inhibition of in vitro invasion was obtained using a specific metalloproteinase inhibitor. Overexpression by transformed cells of a metalloproteinase was detected by gelatin zymography. Together these results suggest that the increased invasive capacity of ras-transformed breast epithelial cells may be largely due to increased metalloproteinase activity.
Thesis (Ph.D.)-University of Natal, Pietermaritzburg , 1996.

Chronic obstructive pulmonary disease (COPD) and lung cancer (LC) are two complex disorders, currently representing the 4th cause of death and the most lethal cancer in Western countries, respectively. A mechanistic link between COPD and LC has been proposed due to an overlap of risk factors of both diseases, where uncontrolled proteolysis may be a critical event in their progress and outcomes. The activity of proteases, their substrates and inhibitors have a significant impact in the extracellular matrix (ECM) remodeling, which may ultimately lead to the development of COPD and LC. Despite the identification of several susceptibility factors in both diseases, there is still many aspects of their pathogenesis that require further elucidation. To address this issue, for our study, we selected 73 proteolysis genes, based on their roles in ECM remodeling, lung expression and/or presence in lung samples and former reports by Genome Wide Association Studies. In a first analysis, we took benefit of The Cancer Genome Atlas on-line database regarding clinical, epidemiological and mutational (somatic and germline) information for two common LC subtypes (adenocarcinoma and squamous cell carcinoma). We found that somatic mutability differs from germline trends and between the two LC subtypes, possibly affecting ECM in distinct ways. Then, we screened by means of PCR-based and Sanger sequencing techniques SERPINB3/B4 and CTSG genes, in a small cohort of COPD and LC patients from which blood and bronchoalveolar lavage fluid samples were collected. Even though, we could not detect any somatic mutation in our sample, for SERPINB3 we detect a considerable number of low-frequency variants in COPD cases in particular, suggesting a misfunction of this gene as a possible genetic risk factor for lung disease. Additional studies in larger cohorts of patients and controls are necessary to confirm this hypothesis.

The extracellular matrix within connective tissues represents a structural scaffold as well as a barrier for motile cells, such as invading tumor cells or passenger leukocytes. It remains unclear how different cell types utilize matrix-degrading enzymes for proteolytic migration strategies and, on the other hand, non-proteolytic strategies to overcome 3D fibrillar matrix networks. To monitor cell migration, a 3D collagen model in vitro or the mouse dermis in vivo were used, in combination with time-lapse video-, confocal- or intravital multiphoton-microscopy, and computer-assisted cell tracking. Expression of proteases, including several MMPs, ADAMs, serine proteases and cathepsins, was shown by flow cytometry, Western blot, zymography, and RT-PCR. Protease activity by migrating HT-1080 fibrosarcoma cells resulting in collagenolysis in situ and generation of tube-like matrix defects was detected by three newly developed techniques:(i) quantitative FITC-release from FITC-labelled collagen, (ii) structural alteration of the pyhsical matrix structure (macroscopically and microscopically), and (iii) the visualization of focal in situ cleavage of individual collagen fibers. The results show that highly invasive ollagenolytic cells utilized a spindle-shaped "mesenchymal" migration strategy, which involved beta1 integrindependent interaction with fibers, coclustering of beta1 integrins and matrix metalloproteinases (MMPs) at fiber bundling sites, and the proteolytic generation of a tube-like matrix-defect by MMPs and additional proteases. In contrast to tumor cells, activated T cells migrated through the collagen fiber network by flexible "amoeboid" crawling including a roundish, elliptoid shape and morphological adaptation along collagen fibers, which was independent of collagenase function and fiber degradation. Abrogation of collagenolysis in tumor cells was achieved by a cocktail of broad-spectrum protease inhibitors at non-toxic conditions blocking collagenolysis by up to 95%. While in T cells protease inhibition induced neither morphodynamic changes nor reduced migration rates, in tumor cells a time-dependent conversion was obtained from proteolytic mesenchymal to non-proteolytic amoeboid migration in collagen lattices in vitro as well as the mouse dermis in vivo monitored by intravital microscopy. Tumor cells vigorously squeezed through matrix gaps and formed constriction rings in regions of narrow space, while the matrix structure remained intact. MMPs were excluded from fiber binding sites and beta1 integrin distribution was non-clustered linear. Besides for fibrosarcoma cells, this mesenchymal-toameboid transition (MAT) was confirmed for epithelial MDA-MB-231 breast carcinoma cells. In conclusion, cells of different origin exhibit significant diversity as well as plasticity of protease function in migration. In tumor cells, MAT could respresent a functionally important cellular and molecular escape pathway in tumor invasion and migration
Die extrazelluläre Matrix (EZM) des Bindegewebes stellt sowohl ein strukturelles Gerüst als auch eine Barriere für migrierende Zellen dar, wie z.B. invadierende Tumorzellen oder zirkulierende Leukozyten. Es ist bisher unklar, wie diese verschiedenen Zelltypen matrix-degradierende Enzyme für eine proteolytische Migrationsstrategie benutzen bzw. ob und wie sie ohne deren Hilfe durch das Gewebe gelangen. Um Zellmigration in EZM zu untersuchen, wurde ein dreidimensionales Kollagenmodell in vitro wie auch Maus-Dermis in vivo eingesetzt und Zellmigration mittels Zeitraffer-Video-, Konfokal- und Multiphoton-Mikroskopie sowie computer-gestützter Zelltracking-Analyse dargestellt. Expression von Proteasen verschiedener Klassen, wie der MMPs, ADAMs, Serinproteasen und Cathepsine, wurde mittels Durchfluss-Zytometrie, Western blot, Zymographie oder RT-PCR detektiert. Gegen Kollagen gerichtete zelluläre Protease-Aktivität wurde mit Hilfe drei neu entwickelter Techniken dargestellt: (i)quantitative Messung von löslichem FITC aus FITC-markiertem fibrillären Kollagen, (ii) mikro-und makroskopische Reorganisation der physikalischen Matrix-Struktur, und (iii) Visualisierung der Topologie fokaler Degradation von Matrixfasern. Die Ergebnisse zeigen, dass hochinvasive spindelförmige HT-1080 Fibrosarkomzellen eine sogenannte "mesenchymale" Migrationsstrategie mit folgenden Charakteristika entwickelten: (i) beta1 Integrin-abhängige Interaktion mit Kollagenfasern, (ii) das "Co-clustering" von beta1 Integrinen und Matrix-Metalloproteinasen an Faserzugstellen und (iii) eine röhrenförmige, durch Proteasen verursachte Matrixdefektbildung. Im Gegensatz zu proteolytischen Tumorzellen migrierten T-Zellen rundlich-elliptoid mittels flexibler Morphodynamik, ähnlich wie Amöben, durch das Kollagennetzwerk und orientierten sich entlang Kollagenfasern, wobei sie keine biochemisch und strukturell detektierbare Faserdegradation zeigten. Um Tumorzell-vermittelte Kollagenolyse zu hemmen, wurde ein Cocktail, bestehend aus Breitspektrum-Protease-Inhibitioren, etabliert, der die Kollagenolyse unter nicht-toxischen Bedingungen um bis zu 98% blockierte. Während in T-Zellen keine morphodynamischen Veränderungen detektiert wurden, entwickelten Tumorzellen eine Verschiebung von proteolytisch mesenchymaler zu unverminderter nicht-proteolytisch amöboider Migration (mesenchymale-amöboide Transition - MAT) aus, sowohl in Kollagenmatrices in vitro als auch in Maus-Dermis in vivo, dargestellt mittels Intravital-Multiphoton-Mikroskopie. Die Tumorzellen "quetschten" sich dabei durch Lücken in der Matrix und bildeten sogenannte Konstriktionsringe aus, während die Matrixstruktur intakt blieb. MMPs lokalisierten nicht mehr an Faser-Kontakstellen auf der Zelloberfläche, und beta1 Integrine lagen nicht mehr geclustert vor. Neben HT-1080 Fibrosarkomzellen wurde MAT auch für MDA-MB-231 Brustkrebszellen epithelialer Herkunft nach Protease-Blockade detektiert. Somit entwickeln migrierende Zellen verschiedener Herkunft eine signifikante Diversität wie auch Plastizität bei der Migration durch EZM aus, resultierend aus der Funktionalität von Matrix-Proteasen. In Tumorzellen könnte MAT einen funktionell wichtigen zellulären und molekularen Anpassungsmechanismus für die Tumorinvasion und -migration darstellen

A novel calmodulin (CaM)-binding cysteine-rich protein from Dictyostelium, cyrA, with epidermal growth factor-like (EGFL) repeats was discovered and characterized. Calcium-dependent and –independent CaM-binding was verified. Western blots show that full length cyrA is detected constitutively throughout development. Analyses of the extracellular medium reveal that cyrA is cleaved and that the fragments containing the N-terminus are secreted early in development, while those containing the C-terminus are secreted later. In support of this, GFP and immunohistochemistry studies reveal that cyrA localizes to the endoplasmic reticulum and secretory vesicles of vegetative cells, and to the extracellular matrix (slime sheath) of migrating slugs. The addition of EGFL1 peptides enhanced cell motility and cAMP-mediated chemotaxis. Finally, cyrA cleavage is regulated by extracellular Dictyostelium CaM and by the extracellular EGFL repeats. In total the data suggest that cyrA is a true matricellular protein that mediates cell motility during multicellular development.

Η σεργλυκίνη (SG) είναι η κύρια πρωτεογλυκάνη που εκφράζεται από τα αιμοποιητικά κύτταρα και συμμετέχει στη ρύθμιση διαφόρων παραγόντων που εμπλέκονται στις αντιδράσεις φλεγμονής. Επιπλέον, φαίνεται ότι διαδραματίζει σημαντικό ρόλο στη βιολογία του πολλαπλού μυελώματος αφού αναστέλλει τη δραστικότητα της κλασικής οδού και της λεκτινικής οδού του συμπληρώματος και προάγει την προσκόλληση των μυελωματικών κυττάρων στο κολλαγόνο τύπου Ι. Παράλληλα, η αυξημένη έκφρασή της σχετίζεται με τον επιθετικό φαινότυπο των κυττάρων καρκίνου του ρινοφάρυγγα. Στην παρούσα διατριβή μελετήθηκε η έκφραση της SG σε κακοήθειες. Τα αποτελέσματά μας αναδεικνύουν την έντονη παρουσία της σε συμπαγείς όγκους λόγω της αυξημένης έκφρασή της είτε από τα καρκινικά κύτταρα είτε από τα κύτταρα φλεγμονής και τα κύτταρα του στρώματος τα οποία επιστρατεύονται κατά την ανάπτυξη του όγκου. Επιπλέον, η αυξημένη έκφρασή της και η έκκρισή της σχετίσθηκαν με το μεταστατικό δυναμικό των κυτταρικών σειρών διαφόρων τύπων καρκίνου. Παράλληλα, ταυτοποιήθηκε η έκφραση του μεταγράφου της SG που προκύπτει από εναλλακτικό μάτισμα με απαλοιφή του εξωνίου 2. Επιπροσθέτως, μελετήθηκε ο βιολογικός ρόλος της SG σε επιθετικά καρκινικά κύτταρα μαστού. Βρέθηκε ότι η SG αλληλεπιδρά με πρωτεΐνες που διαδραματίζουν σημαντικό ρόλο σε κυτταρικές λειτουργίες όπως η αναδιοργάνωση του κυτταροσκελετού, η μεταφορά και ανακύκλωση μορίων από και προς την κυτταρική επιφάνεια και η γονιδιακή ρύθμιση. Η ίδια πρωτεογλυκάνη εκκρίνεται ιδιοσυστατικά από αυτά τα κύτταρα. Τόσο η γλυκοζαμινογλυκανική της σύσταση όσο και η ανασταλτική της δράση έναντι της ενεργοποίησης του συστήματος του συμπληρώματος είναι παρόμοιες με τη SG που εκκρίνεται από τα μυελωματικά κύτταρα. Η συμβολή της SG στον επιθετικό φαινότυπο των καρκινικών κυττάρων επιβεβαιώθηκε με την υπερέκφρασή της σε χαμηλής επιθετικότητας κύτταρα καρκίνου του μαστού. Λειτουργίες όπως ο κυτταρικός πολλαπλασιασμός, η μετανάστευση και η διήθηση των καρκινικών κυττάρων σχετίσθηκαν θετικά με την αυξημένη έκφραση και έκκριση της SG από αυτά τα κύτταρα. Οι επαγωγικές της ιδιότητες καταργήθηκαν με την απαλοιφή των θέσεων πρόσδεσης των γλυκοζαμινογλυκανών από τον πρωτεϊνικό της κορμό. Επιπλέον, διερευνήθηκε το πρωτεολυτικό δυναμικό αυτών των κυττάρων με τη μελέτη της έκφρασης πρωτεολυτικών ενζύμων του εξωκυττάριου χώρου, όπως οι tPA, uPA, MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP και του αναστολέα των μεταλλοπρωτεϊνασών TIMP-1. Παρουσιάσθηκε ότι η υπερέκφραση του πλήρους μορίου της SG αλλά και της μη γλυκοζυλιωμένης της μορφής οδηγούν στη διαφοροποίηση της έκφρασης και της ενεργότητας των μορίων αυτών με τρόπο που ποικίλει ανάλογα με την κυτταρική πυκνότητα. Παρόλα αυτά, τα δεδομένα που προέκυψαν από τη μελέτη της έκφρασης του uPA και της MT1-MMP συσχέτισαν την αυξημένη τους ενεργότητα με την υπερέκφραση της γλυκοζυλιωμένης μορφής της SG και ανέδειξαν την πιθανή συμβολή τους στην επιθετικότητα των καρκινικών κυττάρων μαστού. Συμπερασματικά, η παρουσία της SG είναι έντονη σε πληθώρα συμπαγών όγκων και καρκινικών κυτταρικών σειρών και φαίνεται να σχετίζεται με το μεταστατικό δυναμικό των κυττάρων. Η συμβολή της στον επιθετικό φαινότυπο των καρκινικών κυττάρων περιλαμβάνει τόσο ενδοκυττάριες όσο και εξωκυττάριες δράσεις που μεσολαβούνται από τη γλυκοζυλιωμένη της μορφή.
Serglycin (SG) is the major proteoglycan of hematopoietic origin cells and contributes to the regulation of several inflammatory proteins. Moreover, SG has a significant role in the biology of Multiple Myeloma; It inhibits the activation of the classical and the lectin pathway of complement system. Enhanced SG expression is correlated with the aggressive phenotype of nasopharyngeal cancer cells. In the present study, the expression of SG in several malignancies was investigated. The strong presence of SG in solid tumors due to its elevated expression either by cancer cells or by inflammatory and stomal cells was revealed. Furthermore, SG elevated expression and secretion was correlated with the high metastatic potential of several cancer cell lines. The expression of the alternatively spliced SG mRNA (variant 2) was identified. This variant lacks exon 2. The role of SG in the biology of aggressive breast cancer cells was studied. SG interacts with significant mediators of actin cytoskeleton reorganization, protein transport and regulation of gene expression. This proteoglycan is constitutively secreted by aggressive breast cancer cells and shares the same glycosaminoglycan (GAG) moieties and inhibitory effects torward complement system activation as the secreted SG by myeloma cells. SG contribution in the aggressive phenotype of cancer cells was studied via the overexpression of the moleclule in the low aggressive breast cancer cells. Cellular functions such as proliferation, migration and invasion of cancer cells were positively correlated with the elevated expression and secretion of SG. These properties were abolished by the deletion of GAG binding sites from SG core protein. Moreover, the proteolytic potential of the overexpressing cells was examined via the expression of ECM degrading molecules, such as tPA, uPA, MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP, and TIMP-1 MMP inhibitor. Altered expression and activity rates of these enzymes correlated with the overexpression of either the full length or the truncated form of SG in a manner which depends on the cellular density. Interestingly, enhanced MT1-MMP activity followed only the overexpression of the full length molecule indicating the contribution of this MMP in breast cancer cell aggressiveness. The data above revealed the intense presence of SG in several solid tumors and cancer cell lines and the correlation of SG expression with the metastatic potential of cancer cells. Its contribution to cancer cells aggressive phenotype includes both intracellular and extracellular functions which are mediated by the glycanated form of SG.