Atherosclerosis is an inflammatory disease of the cardiovascular system. Discoidin domain receptor 1 is a receptor tyrosine kinase that binds collagens. Previous work in our lab has shown that deleting DDR1 in a mouse model results in attenuation of atherosclerosis, with fewer macrophages in the plaque. The aim of this study was to determine what changes in macrophage behaviour due to the lack of DDR1 was attenuating plaque development. In order to carry out experiments, primary mouse peritoneal macrophages were used. DDR1-deficient macrophages adhered significantly less to type IV collagen and fibronectin compared to DDR1-expressing cells. In addition, when plated on type IV collagen and fibronectin, DDR1-deficient macrophages produced decreased levels of MCP-1 protein, a cytokine known to be important in plaque development, particularly in leukocyte recruitment to plaque. These results suggest that DDR1 is an important mediator in macrophage adhesion to matrix and macrophage cytokine production

Atherosclerosis is an inflammatory disease of the cardiovascular system. Discoidin domain receptor 1 is a receptor tyrosine kinase that binds collagens. Previous work in our lab has shown that deleting DDR1 in a mouse model results in attenuation of atherosclerosis, with fewer macrophages in the plaque. The aim of this study was to determine what changes in macrophage behaviour due to the lack of DDR1 was attenuating plaque development. In order to carry out experiments, primary mouse peritoneal macrophages were used. DDR1-deficient macrophages adhered significantly less to type IV collagen and fibronectin compared to DDR1-expressing cells. In addition, when plated on type IV collagen and fibronectin, DDR1-deficient macrophages produced decreased levels of MCP-1 protein, a cytokine known to be important in plaque development, particularly in leukocyte recruitment to plaque. These results suggest that DDR1 is an important mediator in macrophage adhesion to matrix and macrophage cytokine production

During development of epithelial organs, epithelial cells collectively migrate and invade into their surroundings to form complex 3D structures, such as the tubular ducts and alveoli of the mammary gland. A prerequisite for coordinating such collective movement is apicobasal cell polarity. This polarity divides the plasma membrane of epithelial cells into an apical domain towards the lumen of structures and a basal domain facing the matrix. Polarity is essential for the functionality of epithelial tissues and signals from the surrounding matrix are known to participate in its establishment. In contrast, loss of polarity has been associated with progression of diseases such as cancer. During epithelial tubulogenesis, the pro-invasive enzyme MT1-MMP is regulated according to apicobasal polarity. This regulation is essential for tubulogenesis to occur and restricts MT1-MMP activity to the tip of protruding tubules by ensuring that the enzyme only localises to the basal, matrix-abutting cell surface in this location. Signals from fibrillar collagen I contribute to regulating the polarised distribution of MT1-MMP. How such signals are transmitted and influence polarised trafficking is however not understood. In this study, I found that inhibition of the collagen receptor DDR1 disturbed the apicobasal distribution of MT1-MMP in MDCK cells. In 3D environments, DDR1 inhibition blocked MT1-MMP-dependent tubulogenesis of epithelial cells, which instead formed compact, multi-layered aggregates. Furthermore, polarisation of the epithelial cell membrane into an apical and a basal domain failed in absence of DDR1 signalling, suggesting that DDR1 affects establishment of epithelial polarity. In support of this, the effects of DDR1 signalling on apicobasal polarity were not limited to MT1-MMP- dependent morphogenesis, but also proved essential for polarisation of cells during 3D morphogenesis that did not require ECM degradation. An investigation of signalling downstream of DDR1 in establishment of apicobasal polarity revealed this to involve modulation of cytoskeletal tension. Inhibition of DDR1 in 2D culture of MDCK cells thus increased ROCK-dependent phosphorylation of MLC along cell-cell junctions, suggesting that DDR1 can suppress ROCK activity. Importantly, the ROCK-suppressing function of DDR1 contributed to establishment of polarity in MDCK cells in 3D matrices, where inhibition of ROCK activity rescued the formation of polarised cysts in absence of DDR1 signalling. The role of DDR1 in epithelial organisation was reflected in the epithelium of the mammary gland of lactating DDR1-null mice, which had smaller alveoli with a diffuse distribution of basement membrane components compared to wild type mice. Furthermore, DDR1 inhibition attenuated formation of milk-producing mammospheres during lactogenic differentiation of mammary epithelial cells in vitro in a ROCK- dependent manner. This suggests that the ROCK-suppressing function of DDR1 observed in MDCK cells is important for morphogenesis of other epithelial cell types as well. Overall, this study suggests that DDR1 signalling contributes to epithelial polarisation and morphogenesis in a manner involving regulation of cytoskeletal organisation, at least partly through regulation of ROCK activity.

S'ha demostrat que la mielina està alterada en el cervell de pacients amb esquizofrènia. El gen domini discoidina 1 (DDR1), que s'expressa en mielina, s'ha trobat associat a esquizofrènia. Ambdues troballes fetes al grup de recerca on he desenvolupat el treball de tesi doctoral.· Contribucions i coneixements nous que aporta la tesiEn síntesi podem afirmar que DDR1 és clau en el procés de mielinització. Una regulació a la baixa d'aquest gen podria estar associat a malalties de mielina.Estudi1.Caracterització conductual durant el segon període de remielinització de ratolins tractats amb cuprizona al 0.2% durant 6 setmanes.-Pèrdua de la compactació i síntesi de la mielina en el procés de desmielinització.-Increment de la compactació i síntesi de la mielina durant el pic de remielinització.1. Alteracions estructurals dels mitocondris pel tractament de la cuprizona.2. Alteracions en el comportament.a. Període d'exposició a la cuprizona: menor activitat del SNC, deteriorament de les funcions motores i menys ansietat.b. Període de recuperació: major activitat del SNC, deteriorament de les funcions motores, menys ansietat i una menor activitat exploratòria. Estudi 2. Estudi 2. Caracterització conductal i estructural de la mielina en el ratolí deficient per DDR1 i estudi de la seva capacitat de remielinització. D'aquest estudi es desprenen els següents resultats:1. Alteracions en l'àrea, la densitat de l'axó i el gruix de la beina de mielina.2. Disminució del potencial remielinitzador.Alteracions en el comportament: disfuncions motores, hiporeactivitat del SNC, increment de l'ansietat i de l'activitat exploratòria. Els efectes de la cuprizona accentuen el comportament ansiós d'aquests animals.Estudi 3. El receptor domini discoidina 1 es troba sobre expressat durant la diferenciació de la línia cel·lular oligodendroglial HOG16.-Augment de l'expressió del gen DDR1 paral·lel a l'augment de gens de la mielina.· Metodologia emprada i conclusions més rellevantsLa present tesi doctoral es centra en dos models per aprofundir en el coneixement del paper de DDR1 en la mielina. Per una banda s'ha estudiat l'expressió de DDR1 en línies cel·lulars oligodendroglials sotmeses a diferenciació. Per altra banda s'ha caracteritzat tant histològicament com funcionalment el ratolí deficient en DDR1. El treball es divideix en tres estudis.Estudi 1. Caracterització conductual durant el segon període de remielinització de ratolins tractats amb cuprizona al 0.2% durant 6 setmanes. Desenvolupat a la Facultat de Medicina i Ciències de la Salut. Estudi descriptiu dels canvis ultraestructurals i de comportament dels ratolins alimentats amb una dieta 0.2% v/v de cuprizona durant 6 setmanes.Estudi 2. Caracterització conductal i estructural de la mielina en el ratolí deficient per DDR1 i estudi de la seva capacitat de remielinització. Desenvolupat a la Facultat de Medicina i Ciències de la Salut i a la Universitat de Toronto, Canadà. Estudi descriptiu del ratolí deficient en DDR1 i resposta a la demielinització per tractament amb cuprizona Estudi 3. El receptor domini discoidina 1 es troba sobre expressat durant la diferenciació de la línia cel·lular oligodendroglial HOG16. Desenvolupat a la Facultat de Medicina i Ciències de la Salut. Estudi in vitro de diferenciació del llinatge cel·lular oligodendroglial HOG16.· Resultats tangibles de la investigació: llibres, articles, comunicacions a congressos,...1. Franco-Pons N, Tomàs J, Roig B, Auladell C, Martorell L, Vilella E. Discoidin domain receptor 1, a tyrosine kinase receptor, is upregulated in an experimental model of remyelination and during oligodendrocyte differentiation in vitro. J Mol Neurosci. 2009 May;38(1):2-11. Epub 2008 Oct 4. PubMed PMID: 18836851. 2. Tomas-Roig J, Torrente M, Roig B, Moyano S, Vogel W, Colomina T, Vilella E. Brain deficits and impaired experimentally-induced remyelination in disocidin domain receptor 1 null mice. Enviat per a la seva publicació. * Estades de la persona que es doctora a altres grups de recercaL'estudi del model de desmielielinització i remielinització induït per cuprizona al 0.2% en ratolins deficients pel gen DDR1 ha tingut lloc amb la col·laboració i participació del doctorant en el grup de recerca encapçalat pel Dr. Wolfgang F. Vogel (Universitat de Toronto, Canadà) * Altres comentaris Participació en articles relacionats:Roig B, Franco-Pons N, Martorell L, Tomàs J, Vogel WF, Vilella E. Expressionof the tyrosine kinase discoidin domain receptor 1 (DDR1) in human centralnervous system myelin. Brain Res. 2010 Apr 6. [Epub ahead of print] PubMed PMID: 20380825. / We have demonstrated that myelin is deteriorated in schizophrenic patients. Discoidin domain 1 gene (DDR1) is expressed in myelin and it's also associated with schizophrenia. Both findings were made by our research group.· Contributions and knowledge provided by the thesisIn summary we can say that DDR1 is a key process of myelinazation. A down regulation of this gene might be associated with diseases of myelin.Study 1. It has been described a loss of myelin compaction and synthesis during the demyelination process. It has carried out an increase of compaction and synthesis of myelin during remielinization peak.1. Structural alterations of mitochondria for the treatment of cuprizona.2. Alterations in behavior.a. Period of exposure to cuprizone: less activity of the CNS, impairment function motor and less anxiety.b. Recovery period: increased activity of the CNS, impaired motor functions, less anxiety and less exploratory activity.3. mRNA analysis have showed an increase of DDR1 expression during the remyelinization peak in parallel to another myelin gene.Study 2. From this study derived the following results:1. Alterations in the area, the density of the axon and the thickness of the myelin sheath.2. Decrease of the remyelinization potential. Alterations in behaviour: motor dysfunctions, hiporeactivity of the CNS, increase of anxiety and exploratory activity. Cuprizone accentuates the effects of anxious behaviour of these animals.Study 3.We described an increased expression of DDR1gene parallel to myelin genes.· Methodology used and most important conclusions This thesis focuses on two models to improve our knowledge of the role of DDR1 in myelinization. On one hand we have studied the expression of DDR1 in oligodendroglial cell lines undergoing differentiation. On the other hand has been characterized both histologically and functionally DDR1deficient mouse. The work was divided into three studies. Study 1. Behavioural features and ultrastructural myelin characterization during the second remyelinization period in those mice treated with cuprizone 0.2% for 6 weeks. It has been developed at the Faculty of Medicine and Health Sciences (Reus, Spain). Study 2. Behavioural characterization and myelin structure in knock-out mice for DDR1 and study its ability to carry out the remyelinization. It has been developed at the Faculty of Medicine and Health Sciences (Reus, Spain) and at the University of Toronto (Toronto, Canada). Descriptive study of DDR1 knock-out mice and their response to cuprizone treatment. Study 3. The discoidin domain receptor 1 is expressed during differentiation of the oligodendroglial cell line HOG16. It has been developed at the Faculty of Medicine and Health Sciences. In vitro study to evaluate changes in mRNA expression and changes in cells morphology. · Published papers:1. Franco-Pons N, Tomàs J, Roig B, Auladell C, Martorell L, Vilella E. Discoidin domain receptor 1, a tyrosine kinase receptor, is upregulated in an experimental model of remyelination and during oligodendrocyte differentiation in vitro. J Mol Neurosci. 2009 May;38(1):2-11. Epub 2008 Oct 4. PubMed PMID: 18836851. 2. Tomas-Roig J, Torrente M, Roig B, Moyano S, Vogel W, Colomina T, Vilella E. Brain deficits and impaired experimentally-induced remyelination in disocidin domain receptor 1 null mice. Submitted for its publication.· Stays in other countriesThe study of demyelinization and remyelinization model induced by cuprizone 0.2% on knock-out DDR1 mice has taken place with the collaboration and participation of the PhD student in the research group led by Dr. Wolfgang F. Vogel (University of Toronto, Canada). · Other commentsParticipation in related papers:Roig B, Franco-Pons N, Martorell L, Tomàs J, Vogel WF, Vilella E. Expression of the tyrosine kinase discoidin domain receptor 1 (DDR1) in human central nervous system myelin. Brain Res. 2010 Apr 6. [Epub ahead of print] PubMed PMID: 20380825.

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Le cancer colorectal (CCR) demeure l'une des principales causes de décès par cancer dans le monde. Récemment, une nouvelle immunothérapie basée sur le ciblage du récepteur à activité tyrosine kinase (RTK) EGFR via l'anticorps cetuximab a montré une amélioration significative de la survie des patients atteints de CCR métastatique (CCRm). Cependant, une grande partie de ces patients présente une résistance qui est associée à la présence de mutations gains de fonction dans la voie de signalisation Ras. Dans ce contexte, l'identification de cibles thérapeutiques Ras-indépendantes pourrait avoir un intérêt particulier. Une analyse pharmacologique effectuée au laboratoire démontre que le nilotinib, un inhibiteur de la TK oncogénique BCR-ABL utilisée en clinique pour le traitement des leucémies myéloïdes chroniques, affecte également les propriétés invasives et métastatiques de cellules de CCR. Nous avons identifié la cible majoritaire de cet inhibiteur dans cette réponse tumorale : il s'agit de DDR1, un récepteur au collagène ayant une activité TK intrinsèque. Mon travail de thèse a mis en évidence que DDR1 est fréquemment et fortement activé dans les métastases de patients atteints de CCR. J'ai ensuite analysé la signalisation DDR1-dépendante dans ces cellules tumorales par phosphoprotéomique quantitative. Cette analyse montre la nature Ras-indépendante de cette signalisation et révèle la protéine de signalisation BCR comme un nouveau substrat essentiel de DDR1 pour induire l'invasion cellulaire. Enfin, je montre que l'inhibition de DDR1 par le nilotinib améliore la réponse au cetuximab y compris pour les cellules tumorales ayant une mutation oncogénique de K-Ras ou B-Raf. En conclusion, ces résultats suggèrent que le ciblage de DDR1 par le nilotinib pourrait avoir un intérêt thérapeutique dans le traitement des CCRm. / Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the Western world. Cancer that metastasizes to distant sites is usually not curable, although chemotherapy can extend survival. Recently, a novel immunotherapy approach based on targeting the EGFR tyrosine kinase (TK) via the antibody cetuximab was shown to significantly improve the survival of patients with metastatic CRC (mCRC). However, only patients with wild-type KRAS may hope to gain from EGFR inhibition. The majority of patients expresses oncogenic K-Ras alleles and thus have hyperactive Ras cascade operating independently from EGFR. Therefore, there is an urgent need for discovery of a new Ras-independent target that regulates tumor cell invasion and metastasis in mCRC. A pharmacological approach in the lab revealed that the small inhibitor of the leukemic TK BCR-Abl also inhibits invasive and metastatic abilities of CRC cells. This effect was also observed in cells expressing deregulated KRAS signalling. We next identified the receptor for collagen and atypical TK DDR1 as that the main target of nilotinib in these cells. Accordingly, DDR1 was found frequently and strongly activated in metastatic nodules of CRC patients. I also characterized DDR1 signalling by quantitative phosphoproteomic. This analysis revealed the Ras-independent nature of DDR1 signalling but also the Rho GTPase regulator BCR as an essential DDR1 substrate that leads to cell invasion. Finally, I demonstrate that nilotinib potentiates cetuximab response in CRC cells expressing oncogenic K-Ras signaling. Overall, these results suggest that the targeting of DDR1 by nilotinib may be of therapeutic value in mCRC.

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L’actine est impliquée dans de nombreuses fonctions cellulaires physiologiques et pathologiques. Au cours de ma thèse j'ai analysé le rôle de l'actine i) lors de l’invasion tumorale et ii) dans la formation des fenêtres des cellules endothéliales sinusoïdales hépatiques. i) Les cellules tumorales forment des structures d’actine permettant la dégradation de la matrice extracellulaire (MEC) nommés invadosomes. Mes travaux ont permis de démontrer que la RhoGTPase Cdc42 régule la formation de la structure d’actine qu’est l’invadosome, tandis que la protéine d’échafaudage Tks5 est requise pour l’activité de dégradation aboutissant à un invadosome fonctionnel. Ces deux molécules constituent la signature moléculaire minimale des invadosomes. Nous avons établi que le collagène de type I qui est surexprimé dans le microenvironnement tumoral induit la formation d’invadosomes linéaires (Lis). Nous avons identifié le récepteur à domaine discoïdine 1 (DDR1) comme spécifiquement responsable de la formation des Lis. Son interaction avec le collagène fibrillaire permet le recrutement du facteur d’échange des RhoGTPases, Tuba et l’activation de la Cdc42 conduisant à la formation d’un Li. DDR1 est impliqué dans l’invasion tumorale et sa surexpression est de mauvais pronostic dans plusieurs cancers comme le poumon ou encore le sein. Le récepteur DDR1 est également impliqué dans la cohésion cellulaire au cours de la migration collective des cellules tumorales. Nous avons démontré que dans un contexte riche en collagène de type I, DDR1 a une double localisation et donc différents rôles associés dans la migration collective. D’une part un rôle de cohésion cellulaire et d’autre part un rôle dans la dégradation de la MEC. Nous tentons de démontrer que ces différentes fonctions impliquent différentes isoformes de DDR1. Nous souhaitons par la suite déterminer les mécanismes moléculaires qui régulent l’expression, la localisation et la signalisation associées aux différentes isoformes de DDR1. ii) Dans un contexte physiologique, les capillaires sanguins du foie présentent des pores transcellulaires ou fenêtres, qui permettent les échanges bidirectionnels entre le sang et les hépatocytes pour assurer la fonction de filtration de cet organe. Au cours du processus de fibrose ces fenêtres sont perdues, réduisant les échanges. Nous avons démontré le caractère réversible de la perte des fenêtres mais aussi que l’actine n’était pas impliquée dans la formation de ces structures. Nous avons développé une méthode de visualisation en microscopie haute résolution STED de ces structures, permettant pour la première fois une analyse sur cellule vivante. Par une approche de spectrométrie de masse couplée à notre nouvelle méthode d’observation en STED, nous voulons valider la co-localisation des fenêtres avec des marqueurs potentiels identifiés. / Actin is involved in many physiological and pathological cellular functions. In my thesis I analyzed the role of actin i) during tumor invasion and ii) in the formation of fenestrae in liver sinusoidal endothelial cells. i) Tumor cells form actin-based structures, invadosomes, involved in extracellular matrix (ECM) degradation. My work has demonstrated that the RhoGTPase Cdc42 regulates the formation of invadosomes, while the Tks5 scaffold protein is required for the matrix degradation activity. These two molecules form a minimum molecular signature for invadosomes. We found that type I collagen, which is overexpressed in the tumor microenvironment, induces the formation of linear invadosomes (Lis). We have identified the discoidin receptor 1 (DDR1) to be specifically responsible for Lis formation. Its interaction with the fibrillar collagen allows the recruitment of GEF Tuba and the activation of Cdc42 RhoGTPase leading to Li formation. DDR1 is implicated in tumor invasion and its overexpression is a poor prognosis in many cancers like lung or breast. The DDR1 receptor is also involved in cell cohesion in the collective migration of tumor cells. We have demonstrated that in a context rich in type I collagen, DDR1 has a dual location and therefore possesses different roles in the collective migration of tumor cells: a role in cell cohesion and a role in ECM degradation. We are analyzing the role of the different DDR1 isoforms in this process. We wish subsequently to determine the molecular mechanisms that regulate the expression, localization and signaling associated with these different isoforms. ii) In a physiological context, the liver capillaries have transcellular pores that allow bidirectional exchanges between the blood and the hepatocytes to ensure the proper filtering function of that organ. During the fibrosis process, these pores are lost thus decreasing the exchanges. We have demonstrated that the loss of these “fenestrae” is reversible and also that actin does not play a role in their formation. We have developed a novel method to analyze these structures in living cells using high resolution STED microscopy. Now, by using mass spectrometry approach coupled to our new observation methods in STED, we want to validate the fenestrae co-localisation with potential markers identified.

Les invadosomes sont des structures d’invasion plastiques et dynamiques qui interagissent avec leur microenvironnement. Ils possèdent différentes fonctions telles que l’adhésion, la mécanotransduction ou encore la dégradation de la matrice extracellulaire (MEC). Mon travail de thèse s’est concentré sur i) l’étude globale de la composition des invadosomes par spectrométrie de masse et ii) sur l’impact d’éléments du microenvironnement dans la formation de ces structures d’invasion.i) Les invadosomes sont des complexes multi-protéiques dont tous les partenaires ne sont pas encore totalement identifiés. Au laboratoire, une nouvelle approche combinant la microdissection laser suivie d’une analyse par spectrométrie de masse, a été développée. Cette technique a été appliquée à l’étude des invadosomes rosettes. Nous avons ainsi mis en évidence une nouvelle fonction associée aux invadosomes, en les définissants comme des sites actifs de traduction protéique. Les invadosomes cependant, sont des structures plastiques dont la formation et la morphologie sont modulées par différents éléments de l’environnement. Nous souhaitons à présent déterminer les partenaires communs et spécifiques entre les différentes organisations des invadosomes afin d’identifier les molécules impliquées dans cette plasticité.ii) La formation des invadosomes peut être induite par différents éléments du microenvironnement comme des facteurs de croissance ou encore la composition et la rigidité de la MEC. Le TGF-β est un facteur de croissance impliqué dans la formation des invadosomes, dans la promotion de la rigidité de la MEC et dans la fibrose hépatique pouvant mener au développement du carcinome hépatocellulaire. Nous avons alors étudié l’impact du TGF-β dans la formation des invadosomes linéaires en contexte de collagène de type I. Nous montrons que le TGF-β module la machinerie moléculaire associée aux invadosomes linéaires en induisant l’expression de DDR1 et MT1-MMP, ainsi que des éléments impliqués dans leur formation tels que le collagène I. Ces modulations sont dépendantes de la voie de signalisation canonique du TGF-β passant par Smad4 et favorisent la formation et l’activité des invadosomes linéaires. De plus, le TGF-β induit une surexpression de la LOXL2 qui est une enzyme de réticulation du collagène, augmentant la rigidité de la matrice ce qui favorise la formation des invadosomes.Les résultats obtenus durant ma thèse auront permis de mieux définir les éléments impliqués dans la composition et la formation des invadosomes. / Invadosomes are plastic and dynamic invasive structures interacting with the microenvironement. Those structures are involved in several functions as adhesion, mecanotransduction and degradation of the extracellular matrix (ECM). My PhD work focuses on i) the study of the invadosomes composition by mass spectrometry and ii) on the impact of microenvironmental elements on the formation of those invasive structures.i) Invadosomes are multi-protein complexes in which all partners are not yet fully identified. In the laboratory, a new approach combining laser micordissection followed by mass spectrometry analysis was developed. This technique has been applied to the study of invadosome rosettes. We have demontrasted a new function associated with indosomes, defining them as active sites of protein translation. Invadosomes, however, are plastic structures whose formation and morphology are modulated by different elements of the environment. We now wish to determine the common and specific partners between the different invadosomes organizations in order to identify the molecules involved in their plasticity.The invadosomes formation can be induced by different elements of the microenvironment such as growth factors or the composition and rigidity of the ECM. TGF-β is a growth factor involved in the invadosomes formation, in the ECM rigidity and in liver fibrosis that can lead to the development of hepatocellular carcinoma. We have studied the impact of TGF-β in the formation of linear invadosomes in the context of type I collagen. We show that TGF-β modulates the molecular machinery associated with linear invadosomes by inducing the expression of DDR1 and MT1-MMP, as well as elements involved in their formation, such as collagen I. These modulations are dependent on the TGF-β canonical signaling pathway through Smad4 and promote the formation and activity of linear invadosomes. In addition, TGF-β induces an overexpression of LOXL2, which is a collagen cross-linking enzyme, increasing the matrix stiffness and promotes the formation of these structures.Taken together, these results enabled us to better define the elements involved in the composition and formation of invadosomes.