The high rate of obesity is having a significant impact on human health. Understanding the underlying biological mechanisms that regulate adipogenesis and adipocyte lipid metabolism is necessary to identify novel approaches that promote weight loss. Conjugated linoleic acid (CLA) is an example of a naturally-derived product reported to exhibit an anti-obesity effect. For this thesis, it was hypothesized that the anti-obesity effects of the t10-c12 CLA isomer is due to lipid droplet dynamics alteration through activation of the Wnt/β-catenin pathway, which leads to weight loss via affecting adipogenesis and/or adipocyte death. Testing of this hypothesis was achieved by examining the effects of the most biologically active CLA isomers, cis-9, trans-11 (c9-t11), trans-10, cis-12 (t10-c12) CLA using in vitro (3T3-L1 cell line) and in vivo (mouse) models. In 3T3-L1 preadipocytes, both c9-t11 and t10-c12 CLA stimulated early stage differentiation, while t10-c12 CLA inhibited late differentiation as indicated by fewer lipid droplets, lower adipokine levels, and decreased levels of perilipin-1 and phospho-perilipin-1 compared to null. The t10-c12 CLA isomer decreased hormone-sensitive lipase (HSL) levels and inhibited lipolysis by activating protein kinase Cα (PKCα). As well, t10-c12-CLA inhibited adipocyte differentiation by stabilizing β-catenin, which sequesters peroxisome proliferator-activated receptor-γ in an inactive complex. Reduced body weight in both db/db and C57B/L6 mice fed t10-c12 CLA was due to less white and brown fat mass without changes in lean body mass or an alteration in feed intake compared to their respective control. t10-c12 CLA did not stimulate cell death in white adipose tissue. Immune cell infiltration was decreased in calorie restricted pair weight control mice, but not with CLA. t10-c12 CLA-induced weight loss did not improve hyperglycemia in db/db mice. In conclusion, the anti-adipogenic effects of t10-c12 CLA in vitro result from stabilization of β-catenin, which alters lipid droplet dynamics through HSL levels and perilipin-1 phosphorylation via the activation of PKCα. In contrast, t10-c12 CLA promotes loss of adipose tissue in vivo, possibly by activating β-catenin, but without influencing either adipogenesis or adipocyte clearance. This study suggests a novel mechanism for the anti-obesity effect of t10-c12 CLA, and highlights the possible side-effects associated with t10-c12 CLA consumption. / February 2016

Beim humanen nichtkleinzelligen Bronchialkarzinom ist die schrittweise Progression vom gutartigen Tumor zur malignen Metastasierung weitestgehend ungeklärt. In einem transgenen Mausmodell für das humane nichtkleinzellige Bronchialkarzinom, in dem in Lungenepithelzellen eine onkogene Mutante der Proteinkinase C-RAF exprimiert wird, können einzelne Schritte im Prozess der malignen Progression entschlüsselt werden. Die durch C-RAF induzierten Adenome zeichnen sich durch eine hohe genomische Stabilität in den Tumorzellen, durch starke interzelluläre Adhäsionskontakte zwischen den Tumorzellen und das Fehlen einer malignen Progression aus. Hier wurde demzufolge untersucht, ob die Auflösung der E-Cadherin-vermittelten Zellkontakte zwischen den einzelnen Tumorzellen eine Metastasierung auslösen könnte. Es wurden zwei genetische Ansätze verfolgt, um die Rolle der Tumorzelladhäsion im C-RAF Modell zu bewerten, die konditionelle Eliminierung des E-Cadheringens Cdh1 sowie die regulierbare transgene Expression von dominant-negativem E-Cadherin. Die Auflösung der E-Cadherin-vermittelten Zelladhäsion führte zur Neubildung von Tumorgefäßen, welche in der frühen Phase der Gefäßbildung durch Wiederherstellung des Zellkontakts reversibel war. Die vaskularisierten Tumore wuchsen schneller, bildeten invasive Fronten aus und führten zur Ausbildung von Mikrometastasen. Es konnte gezeigt werden, dass Beta-Catenin für die Induktion der Angiogenesefaktoren VEGF-A und VEGF-C in Lungentumorzellinien des Menschen und der Maus essentiell war. Lungentumorzellen aus den in situ Tumoren mit aufgelösten E-Cadherin-vermittelten Zellkontakten exprimierten Gene endodermaler und anderer Zellabstammung, was epigenetische Reprogrammierung in Tumorzellen als den Mechanismus bei der malignen Progression vermuten lässt. / Steps involved in the progression of non-small cell lung cancer (NSCLC) to metastasis are poorly understood. Expression of oncogenic C-RAF in lung epithelial cells has yielded a model for non-small cell lung cancer (NSCLC). The induced adenomas are characterised by high genomic stability, a lack of tumor progression and pronounced cell-cell contacts raising the question whether disruption of E-cadherin complexes would promote progression to metastasis. Two genetic approaches were used to evaluate the role of adherens junctions in a C-RAF driven mouse model for NSCLC: conditional ablation of the Cdh1 gene and expression of dominant negative (dn) E-cadherin. Disruption of E-cadherin function caused massive formation of intratumoral vessels that was reversible in the early phase of induction. Vascularized tumors grew more rapidly, developed invasive fronts and gave rise to micrometastasis. ß-catenin was identified as a critical effector of E-cadherin disruption leading to up-regulation of angiogenic inducers (VEGF-A and VEGF-C) in mouse and human lung tumor cell lines. In vivo, lung tumor cells with disrupted E-cadherin expressed ß-catenin target genes of endodermal and other lineages suggesting that reprogramming may be involved in metastatic progression.

Dans les organismes multicellulaires, les cellules génèrent et subissent des forces mécaniques qui se propagent aux cellules voisines. Ces forces peuvent déterminer la forme des tissus et organes, et aussi être converties en signaux biochimiques. Dans un épithélium, les cellules forment un tissu en adhérant directement les unes aux autres grâce à des complexes d’adhérence, tels que les Jonctions Adhérentes. Ces Jonctions Adhérentes sont composées de protéines transmembranaires les E-cadhérines, dont la partie cytoplasmique est sous tension générée par le cytosquelette d’actomyosine par un lien assurée par la β-caténine. La β-caténine est aussi un cofacteur de transcription majeur qui régule l’activité de gènes impliqués dans la transition épithélio-mésenchymateuse une fois dans le noyau. L’accumulation nucléaire et l’activité transcriptionnelle de la β-caténine peuvent avoir lieu à la suite de stimulations mécaniques dans des situations physiologiques et pathologiques, et ont été proposées comme la conséquence d’une libération de la β-caténine des Jonctions Adhérentes suite à sa phosphorylation. Néanmoins, les preuves directes de ce phénomène et ses mécanismes manquent, et le rôle qu’y tient la tension des E-cadhérines n’est pas connu.Dans cette thèse, nous avons établi la relation entre la tension des E-cadhérines et la localisation nucléaire et l’activité de la β-caténine, prouvé l’existence d’une translocation de la membrane au noyau de la β-caténine, et caractérisé les mécanismes moléculaires sous-jacents dans des cellules en migration induite par un facteur de croissance ou par blessure sur un épithélium, deux conditions qui récapitulent au moins partiellement une transition épithélio-mésenchymateuse.Nous avons montré que l’accumulation nucléaire de la β-caténine est due à un départ substantiel de celle-ci de la membrane, spécifiquement dans les cellules en migration. Cette translocation a lieu en aval d’une voie de signalisation impliquant les kinases Src et FAK, et qui conduit à une relaxation de tension des E-cadhérines. Le mécanisme sous-jacent implique une réorganisation du cytosquelette d’actine, caractérisé par un enrichissement des fibres des stress ventrales, soutenant les protrusions, en phospho-myosine, au détriment du cortex d’actine des Jonctions Adhérentes. En revanche, les phosphorylations dans le complexe cadhérine/caténine ne sont pas requises. Ces résultats démontrent que les E-cadhérines ont un rôle de senseur de la mécanique intracellulaire, et que les adhésions focales sont impliquées dans l’activation de la voie de signalisation β-caténine / In multicellular organisms, cells generate and experience mechanical forces that propagate between and within cells. These forces may shape cells, tissues and organs, and also convert into biochemical signals. In a simple epithelium, cells form tissue sheets by directly adhering to one another through adhesion complexes, such as the Adherens Junctions. Adherens Junctions comprise transmembrane proteins E-cadherins, which are under actomyosin-generated tension via a link that contains β-catenin. β-catenin is also a major transcription cofactor that regulates gene activity associated with Epithelial-to-Mesenchyme Transition when translocated in the nucleus. β-catenin nuclear localization and transcriptional activity are mechanically inducible in a variety of healthy and disease models and were proposed to follow phosphorylation-induced -catenin release from E-cadherin. However, direct evidence for this translocation and these mechanisms are lacking, and whether E-cadherin tension is involved is unknown.In this thesis, we assess the relationship between E-cadherin tension and β-catenin nuclear localization and activity, determine the relevance of β-catenin shuttling between membrane and nucleus, and characterize the underlying molecular mechanisms in cells migrating in an at least partial EMT-like fashion upon hepatocyte growth factor (HGF) or wound stimulation. We showed that β-catenin nuclear activity follows a substantial release from the membrane that is specific to migrating cells. This translocation occurs downstream of the Src-FAK pathway, which targets E-cadherin tension relaxation. The underlying mechanisms sufficiently involve actomyosin remodeling, characterized by an enrichment of ventral stress fibers that capture phosphomyosin at the expense of the cortex at Adherens Junctions. In contrast, phosphorylations of the cadherin/catenin complex are not substantially required. These data demonstrate that E-cadherin acts as a sensor of intracellular mechanics in a crosstalk with cell-substrate adhesions that targets β-catenin signaling

Numerous events during development require the tightly controlled and regulated interaction of cells - from gastrulation in the early embryo to axonal pathfinding and remodeling of synaptic networks. Each of these events is dependent upon signals generated by cell-cell interactions, which are in turn specified by a diverse number of cell adhesion molecules. Many families of cell adhesion molecules have been described, and these fall into the broad categories of cadherins, immunoglobulin superfamily (IgSF) members, selectins, and integrins. Activated Leukocyte Cell Adhesion Molecule (ALCAM) is a member of the IgSF, and controls numerous developmental processes, ranging from hematopoiesis to neuronal targeting. Furthermore, this protein has been implicated in the progression of numerous cancers of diverse origins. Despite the variety of developmental and pathological processes in which ALCAM has been implicated, little is known about how it signals in the cell - few extracellular binding partners have been isolated, and, as of this writing, no cytoplasmic interactors have been identified. The purpose of the work presented in this thesis was to elucidate the mechanisms by which ALCAM influences cell behavior, specifically in uveal melanoma cells, and to determine novel extra- and intracellular ligands. Here, I report the regulation of cadherin-based junctions by ALCAM in uveal melanoma cells, as well as provide evidence for a novel extracellular interaction with L1 cell adhesion molecule, and identify three novel intracellular binding partners.

Cell-cell junctions are essential for tissue homeostasis. Prominent among these junctions are adherens junctions and tight junctions. Adherens junctions mediate adhesion between adjacent cells while tight junctions are responsible for establishing apical-basolateral polarity and limiting paracellular permeability. Loss or disruption of either adherens junctions or tight junctions leads to a myriad of disease states, thus these junctions need to be tightly regulated to prevent dysfunction. A unique property of tight junctions is their dependence on adherens junctions for proper assembly and maintenance. Loss or disruption of adherens junction leads to abnormal tight junctions. Understanding the mechanisms that mediate tight junction coupling to adherens junctions is important for treating diseases that arise from disrupted cell-cell junctions. Currently, two controversial models exist for how tight junctions are coupled to adherens junctions. In the first model, the adherens junction protein α-catenin is critical for tight junction assembly. The second model suggests that a second adherens junction protein, nectin is critical for tight junction assembly through binding the tight junction protein ZO-1, and disruption of tight junction assembly is independent of E-cadherin. α-catenin also binds ZO-1, but the consequences of this interaction are unknown. I hypothesized that α-catenin binding to ZO-1 plays a critical role in coupling tight junctions to adherens junctions. To test this, I mapped the ZO-1 binding site on α-catenin and engineered a point mutant of α-catenin that failed to bind ZO-1. Expression of this point mutant in epithelial cells showed that ZO-1 binding to α-catenin is essential for tight junction assembly and maintenance, while adherens junctions were unaffected. These findings established a role for ZO-1 binding to α-catenin in coupling tight junctions to adherens junctions during junction assembly, as well as at steady-state conditions. After discovering the importance of ZO-1 binding to α-catenin in coupling tight junctions to adherens junctions, I wanted to study whether this interaction is critical in a physiological setting. Tight junctions and adherens junctions are both strengthened in response to mechanical force; however the mechanisms responsible for tight junction strengthening were unknown. Using the system I previously developed, I show that ZO-1 binding to α-catenin is essential for increased tight junction integrity in response to mechanical force, coupling changes in tight junctions to increased stability of adherens junctions. Together, these findings identify a novel interaction that is critical for coupling tight junctions to adherens junctions under several conditions, and provide mechanistic insight into the cellular response to mechanical force.

Thermogenic fat is a promising target for new therapies in diabetes and obesity. Understanding how thermogenic fat develops is important to develop rational strategies to treat obesity. Previously, we have shown that Tyk2 and STAT3, part of the JAK-STAT pathway, are necessary for proper development of classical brown fat. Using primary preadipocytes isolated from newborn mice we demonstrate that STAT3 is required for differentiation and robust expression of Uncoupling Protein 1. We also confirm that STAT3 is necessary during the early induction stage of differentiation and is dispensable during the later terminal differentiation stage. Without STAT3, the brown preadipocytes have increased apoptosis early in the terminal differentiation phase. We also show that the block in differentiation is caused by an inability of STAT3 knockouts to down regulate β-catenin by the end of the induction phase. Application of Wnt/β-catenin inhibitors or knockdown of β-catenin during the induction phase is sufficient to fully rescue differentiation of brown adipocytes from the Myf5+ lineage, including reduction in apoptosis, restoration of histone acetylation in the UCP1 promoter and enhancer regions, and full restoration of the expression of brown fat genes. Finally, we show that in the beige lineage, STAT3 is also necessary during the induction phase and can be rescued by Wnt/β-catenin inhibitors, although the rescue is not as robust as it is in the Myf5+ lineage.

Wnt/β-catenin signaling and asymmetric cell division are essential to development and homeostasis in metazoans; these two mechanisms join into one in the Wnt/β-catenin Asymmetry (WβA) pathway in the nematode C. elegans. In WβA, nuclear asymmetry of two β-catenins, SYS-1 and WRM-1, is achieved by two parallel pathways that reduce SYS-1 and WRM-1 levels in the anterior daughter and increase their levels in the posterior daughter. While it is known that many conserved regulators of Wnt signaling are involved in WβA, how these components interact to achieve SYS-1 and WRM-1 asymmetry is not well understood. In this thesis, genetics, transgenics, and live-imaging are used to demonstrate how WβA regulates it’s multiple outputs. It is shown that APR-1/APC and PRY-1/Axin control asymmetric localization of both SYS-1 and WRM-1, and that Wnt signaling explicitly controls APR-1 regulation of either β-catenin via the kinase KIN-19/CKIα. Additionally, it is demonstrated that the Dishevelled proteins DSH-2 and MIG-5 are positive regulators of SYS-1, but negative regulators of WRM-1. Additionally, data from a screen designed to identify novel kinase regulators of Wnt signaling/asymmetric cell division is presented. Overall, this thesis takes current knowledge of conserved Wnt signaling component function and provides a compelling model of how those components are adapted to asymmetric cell division.

Adherens junctions are essential for embryogenesis and tissue homeostasis. The major transmembrane adhesion receptors in adherens junctions are the cadherins, which mediate cell-cell adhesion by binding to cadherins on adjacent cells. Cadherin function is regulated by the protein complexes that assemble at its cytoplasmic tail. Vinculin is one cytoplasmic component of the cadherin adhesion complex, but unlike other junction components, it also is enriched in cell-matrix adhesions. The presence of vinculin in cellmatrix adhesions has commanded the most attention, while little is known about its role in cell-cell adhesions. To define the role of vinculin in adherens junctions, I established a short hairpin RNA-based knockdown/substitution system that perturbs vinculin preferentially at sites of cell-cell adhesion. When this system was applied to epithelial cells, cell morphology was altered, and cell-cell adhesion was reduced owing to a lack of cadherin on the cell surface. I investigated the mechanism for this effect and found that vinculin must bind to beta-catenin to regulate E-cadherin surface expression. Having established a role for vinculin in cell-cell adhesions, the critical question became how vinculin recruitment to and activation at cell-cell junctions are regulated. I found that á-catenin triggers activating vinculin conformational changes. Unlike all of the known vinculin activators in cell-matrix adhesions, alpha-catenin binds and activates vinculin independently of an A50I substitution. Thus, adherens junction activators and cell-matrix activators bind to distinct regions of vinculin to activate this molecule. Using mutant vinculins that cannot be tyrosine phosphorylated, I found that vinculin recruitment to cell-cell adhesions, but not cell-matrix adhesions, requires phosphorylation at Y822. Furthermore, this residue is phosphorylated by Abl tyrosine kinases during the assembly of cell-cell adhesions. Taken together, these studies explain how vinculin is differentially recruited to adherens junctions and cell-matrix adhesions and describes the first known role for vinculin at cell-cell adhesions.

<p>Both Notch and the Wnt pathways are key regulators in maintaining the homeostasis in the intestine. Defects on the key tumor suppressor adenomatous polyposis coli, APC a gene in the Wnt pathway is most frequently mutated in colorectal cancer. Previous studies have indicated that there is a crosstalk between these two pathways. We investigate if there is correlation by first using bioinformatics to find Lef1/Tcf sites in several of the Notch pathway gene promoters. Bioinformatically we found that a lot of the genes contained theses sites controlled by the APC's destruction target β-catenin. By using semi quantitative PCR and western blot we found that Hes 1, Hes 7, JAG 2, MAML 1, Notch 2, NUMB, NUMBL, RFNG and LFNG was downregulated in HT29 colon cancer cells carrying a vector containing wild type APC. All but JAG 2 contains at least one Lef1/Tcf site in their promoter region. The results were verified in HT29 cells transfected with siRNA against β-catenin. We also investigated what would happen to the Lef1/Tcf target gene program of the Wnt pathway, if the Notch pathway was inhibited with the gamma-secretase inhibitor DAPT. Results showed no downregulution of β-catenin or its target gene Cyclin D1.Taken together, these results demonstrate that the Wnt pathway can be placed upstream of the Notch pathway and regulates the latter through β-catenin and the Lef1/Tcf target gene program. However, preliminary results indicate that there is no regulation of APC/β-catenin by the Notch pathway.</p>

The USP4 is a deubiquitinating enzyme found elevated in certain human lung and adrenal tumours. USP4 has a very close relative, USP15, which has caused great difficulty in studying only one or the other. We have had generated two antibodies specific to USP4 and USP15, and have confirmed that the two do not cross react. Although there have been previous findings of interacting partners, possible substrates and pathways in which it is involved, the biological role of USP4 is mostly unknown. We have used these antibodies to determine that USP4 and USP15 expression differs across tissue and cell types, and that expression changes as the organism ages. We have shown that USP4 plays a role in canonical Wnt signaling, perhaps by stabilizing Beta-catenin, and identified GRK2 as a kinase, phosphorylating USP4. These data have provided enough information to form a hypothesis, implicating USP4 with the destruction complex in the Wnt signaling pathway.