Inactivation of p53 by mutation occurs in half of human tumours. The majority of these mutations affect the DNA-binding core domain and hence impair DNA binding and hinder transcription of p53 target genes. A wealth of data indicates that even cancers carrying wild type p53 protein, evolve mechanisms to render the p53 pathway inactive. Thus, inactivation of the p53 response, either by mutation or the alternative mechanisms, allows unpurturbed tumour growth. Recent work identified Anterior Gradient-2 (AGR2) as a protein overexpressed in wild type p53 expressing tumours and it was subsequently shown that AGR2 inhibits p53 pathway. In this study I confirmed that AGR2 protein inhibits p53 and AGR2 depletion potentiates p53-dependent DNA damage response. As there were no physiological signals known that regulate the AGR2-p53 axis, here I set out to identify pathways that activate or inhibit AGR2. I found that transforming growth factor β(TGF- β) triggers AGR2 protein reduction and this is concomitant with the stabilisation and increased activity of p53 protein. TGF-β halts AGR2 transcription in a SMAD4- dependent manner and triggers AGR2 protein degradation involving an ATM kinase. I found that SMAD nuclear interacting protein (SNIP1) mediated the ATMdependent AGR2 degradation. Interestingly, SNIP1 overexpression by itself promoted AGR2 protein degradation. I found that AGR2 protein degradation was proteasome independent and involed autophagy-lysosomal degradation pathway. As the mechanism of p53 inhibition by AGR2 is not known, I reasoned that identifying interactors of AGR2 may potentially further our understanding of the mechanism accounting for AGR2-mediated p53 inhibtion. I isolated the ATP binding protein Reptin in the yeast two-hybrid system and subsequently validated it as an AGR2 binding partner. Mutations of the two ATP binding motifs in Reptin resulted in altered oligomerization and thermostability of Reptin and affected the AGR2-Reptin complex stability. I also identified the Reptin docking site and it was mapped to a divergent octapeptide loop. I found that AGR2-Reptin complex coimmunoprecipitated with the p53 protein. Subsequently, I showed that Reptin protein can influence p53 activity, and depending on local concentration, either inhibit the transcription of p53-genes or chaperone its DNA binding activity. Interestingly, I found that Reptin formed a stable complex, independent of AGR2, with p53 R175H, p53 F270A, p53 S269D and p53 S269A, which has implication for the Reptin function in cancers bearing mutant form of p53 protein.

Intestinal immune cells remain tolerant to the trillions of commensal bacteria present in the gut, with perturbations of this process implicated in development of inflammatory bowel disease (IBD). The cytokine TGF-β is a key factor promoting intestinal immune tolerance, but is secreted in a latent state that requires activation to function. Binding of TGF-β to the integrin αvβ8 is a principal mechanism of TGF-β activation, with mouse models demonstrating a crucial role for αvβ8 expression by dendritic cells and regulatory T cells in intestinal immune regulation. Despite this evidence, very little is known regarding the importance of this activating integrin in human intestinal homeostasis. Utilising flow cytometry here we find that integrin αvβ8 is highly expressed on peripheral blood monocytes with highest levels on intermediate CD14++CD16+ monocytes. Upon monocyte to macrophage differentiation high β8 expression is observed on anti-inflammatory M-CSF differentiated macrophages versus pro-inflammatory GM-CSF macrophages. In monocytes, expression of β8 is upregulated by specific bacterial TLR ligands. Utilising a TGF-β reporter cell line both monocytes and M-CSF MDM display an enhanced ability to activate TGF-β in an αvβ8-dependent manner. Data presented here indicate that macrophage αvβ8-dependent TGF-β activation does not alter expression of surface markers associated with a tolerogenic macrophage phenotype, phagocytosis, or production of the anti-inflammatory cytokine IL-10; nor does TGF-β appear to influence the metabolic profile of macrophages, key differences of which are associated with pro- or anti-inflammatory phenotype. However, the previously undescribed finding of integrin αvβ8 expression on human monocytes and macrophages, which was subsequently confirmed in intestinal populations and found to be downregulated in inflamed IBD mucosa, may highlight an important functional pathway in intestinal immune homeostasis and represent a potential future therapeutic target in IBD.

Les gliomes malins prolifèrent d’une manière très anarchique et elles s’infiltrent dans le parenchyme cérébral. Plusieurs facteurs de croissance, tels que le Transforming Growth Factor Beta (TGF-ß), sont surexprimés dans les gliomes malins. La voie de signalisation du TGF-ß joue un rôle clé dans la régulation des mécanismes liés au métabolisme et à la prolifération cellulaire. L’objectif principal de cette étude est d’évaluer les effets de l’inhibition de la voie de signalisation du TGF-ß sur le phénotype des gliomes malins. Deux méthodes ont été utilisées pour inhiber la voie du TGF-ß. La première est un traitement à la chloroquine, un agent alcalinisant lysosomotropique, qui module à la hausse le pH du Trans Golgi Network (TGN )/endosome. Cet agent inhibe la furine, une protéase nécessaire à la maturation du TGF-ß, ainsi que la métalloprotéases-2 (MMP-2) qui permet la libération du TGF-ß de la matrice extracellulaire (MEC). La deuxième méthode est un traitement avec l’Avasimibe™, un agent qui inhibe l'Acyl-CoA-Acétyltransférase (ACAT) démontrée comme étant activé par le TGF-ß. Les effets de ces deux agents ont été testés sur la sécrétion de TGF-ß, sur le métabolisme et la prolifération cellulaire de la lignée de gliomes murins F98. Par l’immunobuvardage de type Western blot, notre étude a révélé la présence de la forme immature de TGF-ß1 et de Latency Associated Peptide (LAP) dans le surnageant de la lignée de gliomes murins F98. Par le test Enzyme Linked Immunosorbent Assay (ELISA), l’étude a indiqué que la chloroquine et l’Avasimibe™ diminuent de 85 ± 3 % et 65 ± 1 % la concentration de TGF-ß1 mature sécrétée dans le surnageant cellulaire. Les essais WST-1 et BrdU ont montré que des expositions de 24 h et 48 h à la chloroquine et l’Avasimibe™ diminuent significativement de plus de 74 ± 9 % et 86 ± 2 % % le métabolisme et la prolifération cellulaire. La zymographie pousse à croire que les effets de chloroquine étaient associés à une inhibition de 75 ± 1 % de l’activité de la MMP-2 en 48 h. L’ajout de la chloroquine et de l’Avasimibe™ pourrait être bénéfique au traitement standard des gliomes malins en réduisant leur phénotype.

The exogenous expression of master transcription factors (TFs) to drive cell identity changes is an exciting and powerful approach to cell and tissue engineering. Yet, the generation of desired cell types is often plagued by inefficiency and inability to produce mature cell types. Through investigations of the molecular mechanisms of induced pluripotent stem cell (iPSC) generation, I discovered that expression of constitutively active Smad2/3 (Smad2CA/3CA), together with the Yamanaka factors, could dramatically improve the efficiency of reprogramming. Mechanistically, SMAD3 interacted with both co-activators and reprogramming factors, bridging their interaction during reprogramming. Because SMAD2/3 interact with a multitude of master TFs in different cell types, I tested the conversions of B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons. Remarkably, each conversion system was markedly enhanced when the master TFs were co-expressed with Smad3CA. These results revealed the existence of shared molecular mechanisms underlying diverse TF-mediated cellular conversions, and demonstrated SMAD2/3 as a widely applicable cofactor that potentiates the generation of diverse cell types with profound efficiency and maturity.

No description available.

Les cellules NK sont des lymphocytes de l'immunité innée impliqués dans la reconnaissance et l'élimination de cellules tumorales ou infectées par des pathogènes intracellulaires. La biologie des cellules NK est régulée par des facteurs intrinsèques comme les facteurs de transcription ainsi que par des facteurs environnementaux comme les cytokines, produites en condition homéostatique ou inflammatoire. Certaines cytokines, comme l'IL-15, l'IL-12 ou l'IL-18 sont connues pour potentialiser les fonctions effectrices des cellules NK. L'IL-15, en activant la voie STAT5 permet, d'une part, d'assurer la survie des cellules NK et, d'autre part via la kinase mTOR, d'induire leur prolifération, d'augmenter leur métabolisme ainsi que leurs fonctions effectrices. D'autres cytokines comme le TGF-ß sont connues pour inhiber les fonctions des cellules NK. Le TGF-ß1 est une des cytokines les plus immunosuppressives du système immunitaire et, en étant secrété´ par différents types de cancers, il participe a` l'échappement tumoral. Depuis longtemps, les effets du TGF-ß in vitro sont connus pour contrer ceux de l'IL-15. L'objectif de ce travail a été´ d'étudier les effets du TGF-ß sur la biologie des cellules NK. Nous avons observé´ que l'ajout de TGF-ß, in vitro, induit un blocage rapide de l'activation de la voie mTOR par l'IL-15, que le TGF-ß a des effets très proches de ceux de la rapamycine, un inhibiteur spécifique de mTOR et que, in vivo chez la souris, l'activation constitutive des voies de signalisation activées par le TGF-ß induit un phénotype proche de celui de la délétion de mTOR dans les cellules NK / NK cells are innate lymphocytes involved in the recognition and elimination of tumor or infected cells. The biology of NK cells is regulated by intrinsic factors such as transcription factors but also by cytokines produced at steady state or under inflammatory conditions. Some of these cytokines like IL-15, IL-12 or IL-18 are known to increase NK cells functions. IL-15 allows NK cell survival via STAT5 and, via mTOR, increase NK cell proliferation, metabolism and acquisition of functions. In the other hand, cytokines like TGF-ß are known to inhibit NK cell function. TGF-ß1 is a major immunosuppressive cytokine, often secreted by tumor cells and participates to tumor escape. The inhibitory effects of TGF-ß in vitro on IL-2/15 mediated NK cell activation have long been shown, but the mechanism remains unknown. The objective of this work was to characterize the effects of TGF-ß at a molecular level. We have observed that TGF-ß induces a rapid blockade of IL-15 induced mTOR activation, in vitro. TGF-ß and the mTOR inhibitor rapamycin have similar effects. Finally, using genetic mouse models in vivo, constitutive TGF-ß signaling or mTOR deletion results in similar developmental arrests in NK cells

Infection with human gastrointestinal (GI) parasites, such as Trichuris trichiura, affects more than billion people worldwide, causing significant morbidity and health problems especially in poverty-stricken developing countries. Despite extensive research, the mechanisms of induction and regulation of effector immune responses against these parasites are incompletely understood, which hinders the development of anti-parasite therapies. Infection with GI parasite is usually chronic suggesting that parasites are capable of modulating immune responses of their host to prevent expulsion. However, mechanisms by which parasites control host immunity to allow infection are still ill-defined. The aim of this PhD study was to characterise the role of different immunoregulatory mechanisms in immunity to GI parasite infection, with a focus on dendritic cells (DCs), regulatory T cells (Tregs) and the regulatory cytokine transforming growth factor β (TGF-β).Here we showed for the first time that loss of TGF-β-activating integrin alphavβ8 specifically on DCs resulted in protection from chronic infection with Trichuris muris, a mouse model of T. trichiura infection in man. Accelerated expulsion was immune-mediated and although increased levels of protective Th2 cytokines were observed very early during infection, elevated levels of non-protective Th1 cytokines were also detected. Partial depletion of CD4+ or FcεRI+ cells had no effect on the observed phenotype. Since deletion of alphavβ8 on DCs results in decreased numbers of Tregs in the gut, we tested whether depletion of Tregs using a mouse model that allows conditional ablation of Foxp3+ Tregs (DEREG mice) would alter infection development. Although transient Treg depletion at the beginning of infection had no major effect on expulsion kinetics, we observed a tendency for enhanced Th2 responses in DEREG mice. Moreover, even though DC-mediated TGF-β activation via alphavβ8 integrin was essential for T. muris infection development, transient depletion of DCs had no effect on the induction of Th2 responses or parasite expulsion. These data indicate a novel role for the TGF-β-activating integrin alphavβ8 and DCs in regulating effector immune responses during T. muris infection and may contribute to the development of new anti-parasite therapies.

In der vorliegenden Arbeit konnten erstmals der TGF-β-Signalweg und dessen Smad- Signalmoleküle innerhalb der MPCs nachgewiesen werden. Dieser Nachweis erfolgte sowohl auf zellulärer und Gewebeebene als auch auf Gen- und Proteinebene. Zusätzlich konnte auf Gen- und Proteinebene gezeigt werden, dass die Signalmoleküle Smad2, Smad3 und Smad4 in MPCs aus gering erkranktem Meniskusgewebe eine vermehrte Expression aufweisen im Vergleich zu den MPCs aus hochgradig erkranktem Meniskusgewebe. Diese Erkenntnis weist auf eine mögliche protektive Funktion des TGF- β-Signalwegs während degenerativer Prozesse im Meniskusgewebe hin. Um die Effekte des TGF-β-Signalwegs und dessen Smad-Signalmoleküle genauer zu verstehen und besser beschreiben zu können, wurde eine Überexpression der Smad-Signalmoleküle innerhalb von MPCs durchgeführt und die Auswirkungen dieser auf die Kollagen I- und Kollagen II-Synthese genauer beleuchtet. Infolgedessen konnte sowohl eine vermehrte Kollagen I-Synthese als auch eine vermehrte Kollagen II-Synthese festgestellt werden. Dies bestätigt die Annahme, dass dem TGF-β-Signalweg und dessen Smad-Signalmolekülen eine zentrale, protektive Funktion während der Meniskusdegeneration zukommt. Durch die vermehrte Synthese von Matrixkomponenten wird den Degenerationsprozessen innerhalb des Meniskusgewebes entgegengewirkt. Ein nicht degenerierter bzw. ein regenerierter Meniskus besitzt eine biomechanische Schutzfunktion für das Kniegelenk und wirkt somit einer Kniegelenkarthrose entgegen. In Zukunft könnte der TGF-β-Signalweg  einen möglichen Ansatzpunkt für therapeutische Behandlungen bei Meniskusläsionen darstellen. Da Meniskusdefekte häufig direkt mit einer Osteoarthrose im Kniegelenk assoziiert sind, spielt die durch den TGF-β-Signalweg induzierte Regeneration von Meniskusgewebe auch in der Prävention der Osteoarthrose eine zentrale Rolle.

La Bone Morphogenetic Protein-1 (BMP-1) est une métalloprotéase impliquée dans la maturation et l'activation de nombreuses molécules extracellulaires. Parmi celles-ci, on trouve notamment les collagènes fibrillaires, les protéines les plus abondante chez l'Homme, ainsi que les facteurs de croissance de la superfamille du TGF-ß, des protéines pléiotropes. Au travers de ses fonctions, BMP-1 joue un rôle crucial au cours du développement embryonnaire mais également durant les processus physiologiques et pathologiques de remodelage tissulaire (cicatrisation, fibroses, croissance osseuse, cancers...).Le projet présenté dans ce manuscrit a consisté à étudier plusieurs fonctions importantes de BMP-1 au niveau moléculaire et à caractériser les conséquences de ces activités au niveau de plusieurs types cellulaires.Dans un premier temps, un test quantitatif a été mis au point afin de pouvoir étudier en temps réel l'effet de BMP-1 sur les collagènes fibrillaires ainsi que les mécanismes de sa régulation. Par la suite, de nouveaux substrats de BMP-1 ont été mis en évidence, parmi lesquels des co-récepteurs du TGF-ß (Bétaglycan, CD109) ainsi qu'une protéine matricellulaire (TSP-1). L'étude de ces activités a permis de caractériser les multiples voies par lesquelles BMP-1 est capable de réguler l'activité du facteur de croissance TGF-ß.De plus, nous avons mis en évidence que le clivage de ces différents substrats entraine une modulation importante du phénotype de plusieurs lignées cellulaires (HT1080, HEK-293T) avec des effets au niveau de l'adhésion, la prolifération et la migration cellulaire. En conclusion, ce travail révèle que les activités de BMP-1 s'étendent bien au-delà de ce qui est actuellement décrit / Bone Morphogenetic Protein (BMP-1) is a metalloprotease known to be involved in the maturation and activation of several important extracellular proteins, including fibrillar collagens and growth factors of the TGF-beta superfamily. As a consequence, it is essential for embryonic development and tissue remodeling and has been clearly involved in lethal diseases such as fibrosis and cancer.This thesis project focused on the major molecular functions of BMP-1 and their implications for the phenotype of several cell types. First, a quantitative and real-time assay was developed to study the effect of BMP-1 and associated regulatory proteins on fibrillar collagens. Then, new BMP-1 substrates, such as TGF-ß co-receptors (Betaglycan and CD109) and matricellular proteins (TSP-1) were characterized in detail. Especially, we evidenced that these activities played a major role in the regulation of the TGF-ß pathway.Furthermore, we shown that these BMP-1 activities induce major phenotype changes (adhesion, proliferation, migration) in several cell lines including HT1080 and HEK-293T. Altogether, this work reveals that BMP-1 substrates extend far beyond what is presently described and open several perspectives for future studies

Die fortschreitende Vermehrung extrazellulärer Matrixproteine ist zentrales Kennzeichen von chronisch-progressiver Niereninsuffizienz. L-Arginin ist eine semi-essentielle Aminosäure und über seinen endogenen Metaboliten Stickoxid (NO) in komplexer Weise mit renaler Matrixvermehrung verbunden. In dieser Arbeit wurde untersucht, wie sich die Gabe von L-Arginin auf die Matrixexpansion bei experimenteller, immun-vermittelter Nierenerkrankung auswirkt. Im Modell der akuten Anti-Thy1-Glomerulonephritis der Ratte und der chronischen Lupusnephritis der MRL/lpr-Maus wurde gezeigt, daß die Aktivierung des L-Arginin-NO-Stoffwechsels sowohl mit günstigen als auch mit ungünstigen Wirkungen auf die renale Matrixakkumulation verbunden ist. Diese "duale" Wirkung von L-Arginin ist im wesentlichen als Ausdruck der "ambivalenten" Wirkung von NO zu deuten. Antifibrotische Wirkungen von L-Arginin stehen in enger Verbindung mit einer gesteigerten endothelialen NO-Synthese. Neben renaler Blutdrucksenkung vermittelt die endotheliale NO-Synthase auch parakrin wichtige antifibrotische Effekte. Profibrotische Wirkungen von L-Arginin stehen in engem Zusammenhang mit einer gesteigerten NO-Synthese durch induzierbare, destruktive NO-Synthasen. Folgen sind verstärkte Organschädigung und beschleunigte Progression von renaler Funktionseinschränkung. / Ongoing expansion of extracellular matrix proteins is a hallmark of progressive chronic renal insufficiency. L-Arginine is a semi-essential amino acid and alters renal matrix accumulation via its endogenous metabolite nitric oxide (NO) in a complex manner. The present study analyzed how administration of L-arginine affects renal matrix accumulation in experimental immune-mediated disease. In acute anti-Thy1 glomerulonephritis of the rat and chronic lupus nephritis of MRL/lpr-mice, activation of the L-arginine-NO-pathway was related to both beneficial and detrimental actions on renal matrix accumulation. This "dual" effect of L-arginine administration essentially reflects the "ambivalent" nature of NO. Antifibrotic actions of L-arginine are associated with increased endothelial NO synthesis. In addition to lowering glomerular blood pressure, endothelial NO production mediates important paracrine antifibrotic actions. Profibrotic effects of L-arginine are related to increased NO production by inducible, destructive NO synthases, resulting in increased organ damage and accelerated progression of chronic kidney insufficiency.