Background. The Cysteine-rich protein, Connective tissue growth factor, and Nephroblastoma overexpressed protein (CCN) family of matricellular proteins are signaling molecules found in the extracellular space, which can have pro-angiogenic, anti-inflammatory and anti-fibrotic properties. Their expression and role in repair and remodeling after myocardial infarction (MI) remains to be better elucidated. In this study, the age-associated expression of Nov (CCN3) and Wisp1 (CCN4) were examined post-MI in mice. Methods and Results. In vivo, MI was induced in young (6 week) and old (12-14 months) mice. Cardiac function was assessed by echocardiography, showing that LVEF was reduced in old mice (33.9%) at 14 days post-MI compared to young mice (43.9%; p=0.002). RT-qPCR analysis of harvested myocardial tissue revealed that mRNA expression of several matricellular proteins in healthy tissue was decreased by 2.5- to 5-fold in old compared to young mice (p=0.03 for Nov, p=0.04 for Wisp1, p=0.0002 for TnC, p=0.04 for TSP-1). Post-MI, mRNA expression of Nov was reduced in the infarct (by up to 13-fold; p<0.03) and border zone (by up to 16-fold; p<0.002) in old compared to young mice. Nov and Wisp1 protein expression was also reduced in old compared to young mice in the infarct and border zones; specifically, for Nov in the infarct zone (p=0.01) and the border zone (p=0.009) at 2 days post-MI and for Wisp1 in the infarct zone at 2 days (p=0.0003) and 14 days (p=0.003), along with 7 days post-MI in the border zone (p=0.0003). To identify possible sources of matricellular proteins, in vitro culture experiments were performed. The expression of Nov protein was increased (1.9-fold; p=0.006) in TGF-B stimulated cardiac fibroblasts after 48h, as was the expression of the myofibroblast marker a-SMA (1.7-fold; p=0.035). Wisp1 mRNA expression was increased (4.5-fold; p=0.03) in stimulated cardiac fibroblasts after 48h in a hypoxic environment. There was also a trend for increased mRNA expression of Nov (p=0.118) and Wisp1 (p=0.121) in M2 macrophages. Cardiac fibroblasts treated with Nov+TGF-B exhibited greater proliferation (by 29%; p0.01), as did those treated with Wisp1+TGF-B (by 16%; p<0.05). Treatment with Nov or Wisp1 led to an increase in viability of cardiac fibroblasts both in the presence (Nov; p=0.0004, Wisp1; p=0.01) and absence of TGF-B (Nov; p=0.0005, Wisp1; p=0.003). Summary. There is an age-associated difference in the expression of matricellular proteins Nov and Wisp1 between both healthy and MI mice. In vitro studies suggest that cardiac fibroblasts may produce Nov and Wisp1 upon their activation to myofibroblasts. The presence of these proteins was also shown to increase the proliferation and viability of fibroblasts. Therefore, reduced levels of Nov and Wisp1 in old mice may negatively affect the repair and remodeling process post-MI compared to young mice. A better understanding of Nov and Wisp1 function in aging and post-MI repair may help identify novel therapeutic targets for limiting damage post-MI and improving repair and heart function.

Lysophosphatidic acid (LPA) is a potent bioactive lipid component of oxidized low density lipoproteins (oxLDL). High concentrations of LPA have been detected in human atherosclerotic plaques. Our data has shown that LPA highly induces smooth muscle cell (SMC) migration. Cyr61, a matricellular protein, which also accumulates in human atherosclerotic plaques, has been implicated in the injury-induced neointimal formation. Smooth muscle cell migration is a key event in the development of atherosclerosis, and it contributes to the progressive growth of atherosclerotic lesions. Data generated by this study demonstrate that LPA markedly induces Cyr61 expression in mouse aortic smooth muscle cells (MASMC). We hypothesized that LPA-induced matricellular Cyr61 mediates LPA-induced MASMC migration. To date, little is known about the relationship between LPA and Cyr61 in smooth muscle cells; the signaling pathway leading to LPA-induced Cyr61 is unknown. Furthermore, whether Cyr61 contributes to LPA-induced cell migration is unrevealed. Our study demonstrates that LPA, by binding to LPA1 receptor, activates the intracellular signaling pathway leading to the activation of PKCdelta which in turn contributes to the increased expression of Cyr61 in MASMCs. Interestingly, we found that after LPA-induced Cyr61 mRNA has been translated into its protein intracellularly, the de novo synthesized proteins promptly accumulate in the Golgi apparatus and then translocalize to the extracellular matrix. Importantly, our data reveal a novel LPA/Cyr61 pathway in controlling MASMC migration. Understanding the mechanism underlying LPA induction of Cyr61 provides new insight into pathogenesis of atherosclerosis.

Dictyostelium discoideum is a social amoebozoan that is used a model system for studying a variety of cell and developmental processes, especially cell motility and chemotaxis. Genome analyses suggest that this model organism possesses a higher percentage of Epidermal Growth Factor (EGF)-like (EGFL) repeats than any other sequenced eukaryote, including humans. EGFL repeats share strong sequence similarity with EGF. In mammals, EGF binds to an EGF receptor (EGFR) to initiate intracellular signalling that regulates a diversity of cellular processes including cell motility and chemotaxis. Some EGFL repeats, like EGF, have also been shown to increase the rate of cell motility by binding to the EGFR and activating EGFR-dependent signalling. Despite their abundance in Dictyostelium, a function for EGFL repeats in this model eukaryote had not previously been studied. This thesis presents a collection of studies that investigated the function of a specific EGFL repeat from the extracellular, cysteine-rich, calmodulin (CaM)-binding protein CyrA. A synthetic peptide (DdEGFL1), equivalent in sequence to the first 18 amino acids of the first EGFL repeat (EGFL1) of CyrA, was shown to increase random cell motility and cAMP-mediated chemotaxis via a novel signalling pathway that did not require either of the two cAMP receptors that are active during early development of Dictyostelium. Several intracellular signalling components were identified and then incorporated into a model detailing the signal transduction regulating EGFL repeat-enhanced cell movement in Dictyostelium. Finally the expression, secretion, and localization of CyrA are presented to couple the findings from studies on DdEGFL1 function with those for the full-length protein. In mammals, a protein that localizes to the extracellular matrix (ECM) and modulates cellular processes by binding to a cell surface receptor and initiating intracellular signalling is termed a ‘matricellular’ protein. The research presented in this thesis suggests that CyrA is the first matricellular protein identified in Dictyostelium.

Dictyostelium discoideum is a social amoebozoan that is used a model system for studying a variety of cell and developmental processes, especially cell motility and chemotaxis. Genome analyses suggest that this model organism possesses a higher percentage of Epidermal Growth Factor (EGF)-like (EGFL) repeats than any other sequenced eukaryote, including humans. EGFL repeats share strong sequence similarity with EGF. In mammals, EGF binds to an EGF receptor (EGFR) to initiate intracellular signalling that regulates a diversity of cellular processes including cell motility and chemotaxis. Some EGFL repeats, like EGF, have also been shown to increase the rate of cell motility by binding to the EGFR and activating EGFR-dependent signalling. Despite their abundance in Dictyostelium, a function for EGFL repeats in this model eukaryote had not previously been studied. This thesis presents a collection of studies that investigated the function of a specific EGFL repeat from the extracellular, cysteine-rich, calmodulin (CaM)-binding protein CyrA. A synthetic peptide (DdEGFL1), equivalent in sequence to the first 18 amino acids of the first EGFL repeat (EGFL1) of CyrA, was shown to increase random cell motility and cAMP-mediated chemotaxis via a novel signalling pathway that did not require either of the two cAMP receptors that are active during early development of Dictyostelium. Several intracellular signalling components were identified and then incorporated into a model detailing the signal transduction regulating EGFL repeat-enhanced cell movement in Dictyostelium. Finally the expression, secretion, and localization of CyrA are presented to couple the findings from studies on DdEGFL1 function with those for the full-length protein. In mammals, a protein that localizes to the extracellular matrix (ECM) and modulates cellular processes by binding to a cell surface receptor and initiating intracellular signalling is termed a ‘matricellular’ protein. The research presented in this thesis suggests that CyrA is the first matricellular protein identified in Dictyostelium.

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.

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.

Le mélanome est le cancer de la peau le plus dangereux : il est capable de métastaser rapidement vers les ganglions et les viscères, et est réfractaire aux chimio/radiothérapies. De nouvelles thérapies ciblant la kinase BRAFV600 retrouvée dans 60% des mélanomes ont montré des effets spectaculaires en termes de survie globale et sans progression de la maladie. L’efficacité de ces thérapies est compromise par l’apparition fréquente de résistances. Les cellules cancéreuses sont ancrées au sein d’un microenvironnement avec lequel elles interagissent. Elles profitent de facteurs solubles du stroma et de l’adhésion à la matrice extracellulaire (MEC) pour survivre face aux thérapies. La protéine matricellulaire SPARC orchestre les interactions entre les cellules saines ou cancéreuses et leur microenvironnement. Absente dans les mélanocytes, son expression est initiée et augmentée dans les mélanomes, corrélée à la progression tumorale et à un mauvais pronostic clinique. Lorsqu’elle est sécrétée par les cellules de mélanome, elle active la kinase AKT, déstabilise le suppresseur de tumeur p53 et favorise la prolifération/survie tumorale. Nous avons montré que le module SPARC/AKT est un nouveau déterminant de la résistance innée ou acquise des mélanomes mutés BRAFV600 aux anti-BRAF, et mis en évidence l’intérêt du ciblage de SPARC en combinaison avec des anti-BRAF ou -MEK pour optimiser/restaurer la sensibilité des mélanomes à ces inhibiteurs. Nous avons aussi montré que les fibroblastes ganglionnaires ont des caractéristiques de fibroblastes activés, et confèrent une résistance aux anti-BRAF aux cellules de mélanomes en générant une MEC permissive à laquelle elles adhérent. / Melanoma is the most dangerous form of skin cancer due to its high metastatic potential and its resistance to both classical chemo- and radiotherapies. New targeted therapies directed against the V600E oncogenic form of BRAF found in about 60% of patients have demonstrated spectacular efficacy both in terms of progression free and overall survival. However most patients invariably relapse after a few months due to resistance mechanisms. Cancer cells are anchored and interact constantly with their microenvironment. Both soluble factors and the extracellular matrix produced by stromal cells have been shown to contribute to cancer cell resistance to therapies. SPARC is a matricellular protein that orchestrates interactions between normal and/or cancer cells and their microenvironment. While it is absent in melanocytes, SPARC expression increases in melanoma and is correlated with both tumoral progression and a bad prognosis. When SPARC is secreted by melanoma cells, it activates the AKT kinase, destabilizes the p53 tumor suppressor and promotes proliferation and survival. Here we identify the couple SPARC/AKT as a new actor contributing to both innate and acquired resistance to BRAFV600E inhibitors. In addition, we demonstrate that targeting SPARC in melanoma cells increases their sensitivity to both BRAF and MEK inhibitors. Finally we show that lymph node fibroblasts share features of activated fibroblasts and confer resistance to BRAF inhibitors to melanoma cells through the production of a permissive extracellular matrix.

Les proteins matricellulaires (MPs) sont des macromolécules non structurales de la matrice extracellulaire (ECM) qui sont induites de façon transitoire lors du développement, de la réparation et du remodelage tissulaire et lors de l’inflammation. L’expression des MPs peut être déclenchée par des dommages tissulaires aigus, et leur expression à long terme peut contribuer à certaines maladies chroniques. Les MPs agissent principalement pour médier les événements du remodelage tissulaire en facilitant les interactions et les signaux à partir de l’ECM vers l’environnement cellulaire avoisinant. En utilisant des données de RNA-seq provenant de deux modèles distincts de dommages rénaux, soit l’Acide Folique (FA) ou l’Obstruction Urétérale Unilatérale (UUO), nous avons analysé les profils d’expressions de plusieurs familles bien connues de MPs lors des blessures aigues et chroniques. Nous révélons de nouvelles MPs impliquées dans les dommages rénaux et présentons de nouveaux réseaux entre les membres de chaque famille de MPs, en utilisant des outils bioinformatiques. L’expression de l’ARNm de certaines MPs a été confirmée par immuno-buvardage de type Western (WB). Afin d’approfondir notre connaissance des mécanismes de réparation tissulaire et de remodelage de la matrice, nous avons choisi SMOC-2 comme MP modèle dans l’étude des carcinomes cellulaires rénaux (RCC), cancers qui présentent de fortes tendances métastatiques. Nous avons démontré que la surexpression de SMOC-2 ainsi que le traitement avec la protéine recombinante de lignées cellulaires RCC (786-O, et ACHN) induisent un profil métastatique de transition épithélio-mésenchymateuse (EMT) par WB et des tests fonctionnels. Nous avons également démontré que l’inhibition de SMOC-2 par siRNA donne les résultats opposés. L’ensemble de nos travaux utilise la compréhension des patrons d’expressions temporels des MPs pour améliorer notre compréhension des mécanismes et conditions qui supportent une activation persistante dans des états pathologiques chroniques. Globalement, notre étude sur SMOC-2 offre une perspective ainsi qu’un modèle intéressant pour l’étude et la caractérisation de nouvelles MPs dans des maladies impliquant le remodelage et la réparation de la matrice. / Matricellular proteins (MPs) are non-structural ECM macromolecules induced transiently during development, tissue repair and remodeling, and inflammation. Expression of MPs can be triggered by acute tissue injury and their sustained expression can contribute to chronic disease. MPs primarily act to mediate tissue remodeling events by facilitating interactions and signals from the ECM to the surrounding cellular niche. Using published RNA-seq data from two distinct models of kidney injury, Folic Acid (FA) and Unilateral Ureteral Obstruction (UUO), we analyzed the expression profile of various members of well-known MP families during the acute and fibrotic injury phases. We reveal novel MPs implicated in renal injury and present informative networks between members of each MP family using bioinformatic tools. mRNA expression of select candidate MPs were confirmed by Western blot. To extend our understanding of translatable mechanisms in repair and matrix remodeling, we chose SMOC-2 as our MP model to study in Renal Cell Carcinoma (RCC) which has strong metastatic tendencies. SMOC-2 overexpression and recombinant protein treatment of RCC cell lines (786-O, ACHN) were shown to induce a metastatic EMT profile by Western blot analysis, supported by functional assays (proliferation, migration). Silencing SMOC-2 by siRNA showed the contrary results. Taken together, our work utilizes the understanding of temporal expression patterns of MPs to gain insight into mechanisms and conditions that support persistent activation in chronic injury states. Overall, our work with SMOC-2 provides a valuable perspective and template to approach studying and characterizing novel MPs in diseases involving pathological matrix remodeling and repair.