Understanding the biological mechanisms underlying the initiation and progression of breast cancer is an important step for its prevention and treatment. We used an in vitro and in vivo model to demonstrate that p100/p52 and RelB are strongly activated in BRCA1-deficient mouse mammary progenitor cells and human BRCA1-mutation carriers. We found that NF-κB activation induces stem and progenitor cell expansion and inhibits differentiation. Knockdown and pharmacological inhibition showed that the progesterone-independent growth of BRCA1-deficient progenitor cells requires the alternative NF-κB activation mediated by ATM. Remarkably, treatment of mice with the NF-κB inhibitor dimethylaminoparthenolide (DMAPT) resulted in prolonged repression of BRCA1-deficient progenitor cell proliferation, revealing a possible approach to cyclic chemoprevention.

The Inhibitor of Apoptosis (IAP) proteins are a highly conserved group of anti-apoptotic proteins. Cellular IAP 1 and 2 (cIAP1 and 2) are two members of the IAP protein family that regulate the activity of the Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor family. The role and mechanism of the IAPs in ubiquitination are not yet completely understood due to the complexity of this posttranslational modification process. Additionally, The IAPs are involved in a myriad of cellular processes, and many of the process-specific mechanisms by which the IAPs are involved is unknown. I aim to delve deeper into the signalling pathways that are controlled by cIAP1 and cIAP2 by discovering currently unknown protein-protein interactions. In doing so, I will determine which proteins interact with the cIAPs and what signalling pathways these proteins are involved in. Using a BioID approach, I sought to characterize the cIAP1 interactors involved in the canonical and non-canonical NF-κB pathways. I generated a stable cell line expressing TurboID-cIAP1 fusion protein in HEK 293T cells that expressTurboID-cIAP1 at levels comparable to endogenous cIAP1. I identified multiple potential cIAP1 interactors that have ties to the NF-κB pathway. These proteins regulate NF-κB signalling in multiple ways including influencing acetylation and nuclear retention of the NF-κB transcription factors, phosphorylation of NF-κB transcription factors, and RNA splicing of genes involved in the TNFR1 complex I. Further work needs to be done to confirm these interactions and to discover the mechanisms by which these interactions occur. NF-κB signalling is known to have widespread function within the cell and within diseases such as cancer; it will be beneficial to study these interactions to better understand how cancer develops and how to treat it best, especially in patients with a poor prognosis.

L’ubiquitination est une modification posttraductionnelle (MPT) qui gouverne la plupart des processus cellulaires Eucaryotes. Elle consiste en l’ajout d’une ubiquitine via une liaison covalente à une protéine cible, altérant son devenir (activité, localisation, dégradation). Le LUBAC (linear ubiquitin chain assembly complex) est le seul complexe capable de catalyser une forme énigmatique de cette modification, l’ubiquitination linéaire. Ces dernières années, ce complexe ternaire a été identifié comme un acteur majeur dans l’activation de NF-κB en réponse à de nombreux immunorécepteurs. Le but principal de cette thèse est de caractériser la régulation du LUBAC et ses partenaires dans des systèmes variés et d’identifier d’éventuelles MPT en charge de moduler ce complexe. Dans un premier temps, nous avons montré que la sous-unité du LUBAC HOIL1 est dynamiquement clivée par la paracaspase MALT1 en réponse à l’engagement des récepteurs antigéniques dans les lymphocytes pour pleinement activer NF-κB. HOIL1 est également constitutivement clivée dans un sous-groupe agressif de lymphome diffus à grandes cellules B (DLBCL), qui présentent une activation aberrante de MALT1, dévoilant de potentielles stratégies thérapeutiques. Dans un second temps, nous avons observé qu’une partie du partenaire du LUBAC CYLD est liée aux satellites centriolaires, des structures granulaires qui gravitent autour des centrosomes et orchestrent la ciliogenèse. Nos données suggèrent que CYLD contrôle la protéostase des satellites centriolaires et gouverne ainsi la formation du cil primaire. Enfin, nous avons analysé les phases précoces de la nécroptose, une forme de nécrose sous le contrôle du LUBAC. Nous avons mis en évidence que l’activation des canaux Pannexin-1 restreint la production de cytokines pro-inflammatoires associée à la nécroptose. Ensemble, ces travaux illustrent comment les MPT contrôlent les voies de signalisation cellulaires associées à l’ubiquitine et comment elles peuvent être corrompues dans des conditions pathologiques. / Ubiquitination is a pivotal multifaceted post-translational modification (PTM), which governs most cellular processes in Eukaryotic cells. Ubiquitination consists in the covalent binding of ubiquitin onto a target protein, altering its fate (activity, localisation, degradation). The “linear ubiquitin chain assembly complex” (LUBAC) is the only known complex capable of catalysing the enigmatic “head-to-tail” linear ubiquitination. Over the years, this tertiary complex has been shown to be essential for signalling to the NF-κB transcription factors family in response to the stimulation of various immunoreceptors. The primary goal of this PhD was to better understand how the LUBAC and its partners are modulated in different systems and identify possible PTMs regulating this complex. First, we discovered that the LUBAC subunit HOIL1 is dynamically cleaved by the paracaspase MALT1 upon antigen receptor engagement in lymphocytes to further NF- κB activation. HOIL1 is also constitutively cleaved in a subset of aggressive diffuse large B-cell lymphoma (DLBCL), which displays aberrant activation of MALT1, unveiling potential therapeutic strategies. Second, we observed that a portion of the LUBAC’s partner CYLD is bound to the centriolar satellites, a set of granular structures surrounding centrosomes that orchestrate ciliogenesis. Our data suggests that CYLD governs the proteostasis of centriolar satellites, and thereby the formation of primary cilia. Lastly, we analysed the initial steps of necroptosis, a regulated form of necrosis under the control of the LUBAC. We found that activation of Pannexin-1 channels restrains the production of proinflammatory cytokines associated with necroptosis. Altogether, this work illustrates how PTM finely tune ubiquitin-associated signalling pathways, and how they can be perverted in pathological conditions.

L’infection par le VIH-1 est un problème majeur de la santé publique qui touche plus de 35 millions de personnes à l’échelle mondiale. La réplication du VIH-1 est déclenchée par l’activation du promoteur LTR, qui contient deux sites de liaison pour le facteur de transcription NF-κB. Ces sites de liaison sont hautement conservés dans le génome du VIH-1, illustrant ainsi l’importance de NF-κB dans l’activité transcriptionelle des gènes du VIH-1 et la production de nouvelles particules virales. La withaferin A (WA) est une substance bioactive extraite de la plante Withania somnifera, qui possède des propriétés pharmacologiques non négligeables dans la régulation de la réponse immunitaire. Des études récentes ont démontré que le potentiel anti-inflammatoire de la WA est dû principalement à l’inhibition de la voie de NF-κB. Le but de ce projet est de déterminer l’effet de la WA sur la réplication du VIH-1 dans les cellules T, qui sont les cibles principales du virus. Des essais de transfections transitoires de cellules T Jurkat E6.1 avec des plasmides contenant le promoteur du VIH-1 ayant différentes constructions de NF-κB, ont démontré que la WA peut réduire l’activité du promoteur d’une manière dépendante de NF-κB. Quant à la production de particules virales, des essais d’infection avec des virus pseudotypés démontrent que la WA diminue la production virale jusqu’à 90% dans des cellules T stimulées avec PMA/PHA et TNF-α, tandis que les mutants ayant des sites de liaison défective pour NF-κB ne sont pas affectés. Des essais de retardement sur gel ainsi que des immunobuvardages de type Western ont montré que la WA altère l’habilité de NF-κB à transloquer dans le noyau, ce qui se traduit par l’inhibition de la synthèse de l’IκB-α, protéine inhibitrice de NF-κB, phénomène sous contrôle étroite de ce facteur de transcription. Ces résultats suggèrent que la WA pourrait permettre une diminution de la réplication virale d’une manière dépendante de NF-κB et ainsi empêcher la propagation du virus aux cellules T chez les individus infectés.

First published online: 2012-03-10 / 名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成24年4月27日 森谷隆氏の博士論文として提出された

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Nuclear factor kappa B (NF-kB) is a widely expressed family of transcription factors that are involved in a diverse number of processes. These include inflammation or differentiation, survival or apoptosis, and proliferation or cell cycle arrest. NF-kB is usually associated with inhibitory kB proteins (IkB), which mask the nuclear localisation sequence (NLS) of NF-kB and renders it in the cytoplasm. Various stimuli result in the activation of the I kappa B kinase (IKK) protein complex, which phosphorylates IκB proteins and thereby marks them for degradation by the ubiquitin-proteasome pathway. Thereby NF-kB enters the nucleus and acts on its target genes. The study of T- and B-lymphocyte antigen receptor signalling to NF-kB is a field of intense investigation, with much attention being focused on the molecular scaffolding proteins Carma1, Bcl10 and MALT1 and their post-translational modifications. These have been shown to be crucial for the organization of the immunological synapse structure under the activated receptor, to which IKK is recruited and becomes activated, which subsequently leads to the activation of NF-kB. T cell receptor (TCR) activation results in a rapid increase in the intracellular Ca2+ level and NF-kB activation is known to be regulated by those increases, but the mechanisms have remained unclear. Calmodulin (CaM) is a calcium sensory protein that responds to increases in intracellular Ca2+ levels. When CaM binds Ca2+ ions, it leads to structural changes that directly as well as indirectly, through CaM dependent kinases (CaMKs), phosphatases and other enzymes, alters a variety of cellular processes, among them transcriptional regulation. Here CaM is shown to interact directly with Bcl10 in a Ca2+ dependent manner. Increases in the intracellular Ca2+ level are shown to induce the proximity of Bcl10 and CaM in vivo. Carma1 associates with Bcl10 through a CARD-CARD domain interaction that is known to be crucial for TCR signalling to NF-kB. The interaction of CaM with Bcl10 was mapped to the CARD domain and was shown to be a negative regulator for the Bcl10-Carma1 interaction. Inhibition of the CaM interaction by a point mutation within the CaM binding site of Bcl10 results in decreased binding of CaM to Bcl10 in vivo, as well as an increased ability of Bcl10 to induce NF-kB transcriptional activity, which is further enhanced by TCR activating stimuli. NF-kB activation is also shown here to be regulated by CaM indirectly through actions of CaMKII. The CaMKII is recruited to the immunological synapse where it interacts with Bcl10 in an inducible fashion and phosphorylates Bcl10. Phosphorylations of Bcl10 by CaMKII are shown to be important for the ability of Bcl10 to induce NF-κB transcriptional activity. Upon mutation of its most important CaMKII site, Bcl10 fails to activate an NF-kB reporter and an NF-kB target gene (IL-2). This mutated Bcl10 also fails to induce activating phosphorylations of IKKa/b and the kinase JNK2 but not JNK1. Furthermore, phosphorylation of Bcl10 by CaMKII regulates the interactions within the important Carma1, Bcl10, Malt1 signaling complex and the essential signal induced ubiquitinations of Bcl10 and IKKg. Phosphorylation of IKK by TAK1 is also regulated by CaMKII, and serine 82 is a putative CaMKII target site of TAK1 that appears to be important for IκBα degradation. In summary, this thesis explores that not only NF-kB but also CaM is a double-edged sword, since the multi-functional NF-kB family of transcription factors is regulated by CaM both negatively and positively.

Recent evidence suggests that the macrophage scavenger receptor class A (SR-A, aka, CD204) plays a role in the induction of innate immune and inflammatory responses. We investigated whether SR-A will cooperate with Toll-like receptors (TLRs) in response to TLR ligand stimulation. Macrophages (J774/a) were treated with Pam2CSK4, (TLR2 ligand), Polyinosinic:polycytidylic acid (Poly I:C) (TLR3 ligand), and Lipopolysaccharides (LPS) (TLR4 ligand) for 15min in the presence or absence of fucoidan (the SR-A ligand). The levels of phosphorylated IκBα (p-IκBα) were examined by Western blot. We observed that Poly I:C and LPS alone, but not Pam2CSK4 or fucoidan increased the levels of p-IκBα. However, LPS-induced increases in p-IκBα levels were further enhanced when presence of the fucoidan. Immunoprecipitation and double fluorescent staining showed that LPS stimulation promotes SR-A association with TLR4 in the presence of fucoidan. To further confirm our observation, we isolated peritoneal macrophages from SR-A deficient (SR-A-/-), TLR4-/- and wild type (WT) mice, respectively. The peritoneal macrophages were treated with LPS for 15min in the presence and absence of fucoidan. We observed that LPS-stimulated TNFα and IL-1β production was further enhanced in the WT macrophages, but did not in either TLR4-/- or SR-A-/- macrophages, when fucoidan was present. Similarly, in the presence of fucoidan, LPS-induced IκBα phosphorylation, NF-κB binding activity, and association between TLR4 and SR-A were significantly enhanced in WT macrophages compared with LPS stimulation alone. The data suggests that SR-A is needed for LPS-induced inflammatory responses in macrophages.

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling pathway is constitutively active in a variety of cancers, including chronic lymphocytic leukaemia (CLL). The importance of this signalling pathway identifies it as a prime therapeutic target; however, the complexity and potential side effects of inhibiting NF-κB have thus far made the clinical use of NF-κB inhibitors a relatively unexplored resource in this disease. This article discusses the role of NF-κB in CLL as a common crossroad for pathways promoting drug resistance in CLL. We provide the background on how this pathway contributes to both spontaneous and drug-induced apoptosis. Potential new avenues to regulate this pathway in CLL are also discussed.

This study was to examine the effect of estrogen on mechanical stretching-induced cardiac dysfunction in an isolated heart model. The isolated rat hearts were perfused via the Langendorff system and exposed to left ventricular stretching. One group hearts (n = 6) were perfused with 17β-estradiol (100 nM) and the other group hearts (n = 6) were perfused with estrogen plus its receptor antagonist ICI182,780 (1 μM) before myocardial stretching was performed. Control hearts (n = 6) were perfused with perfusion buffer. Cardiac functions were recorded. At the end of perfusion, the hearts were harvested and the levels of tumor necrosis factor-alpha (TNF-alpha), phospho-p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) binding activity were examined. Acute ventricular stretching resulted in significantly decrease in left ventricular developed pressure (LVDP) by 42.7%, maximal positive and negative values of the first derivative of pressure (+dP/dt and -dP/dt) by 43.2%, and 43.5%, respectively. The levels of TNF-alpha, phospho-p38 MAPK and NF-κB DNA binding activity were significantly increased following myocardial stretching. In 17β-estradiol treated hearts, the myocardial functions were significantly improved. The levels of TNF-alpha, phospho-p38 MAPK, and NF-κB binding activity in myocardium were also significantly reduced by 35.7%, 56.9%, and 50%, respectively, compared with untreated stretched hearts. The beneficial effects of 17β-estradiol on the stretched hearts were abolished by ICI182,780. The results suggest that pharmacological dose of 17β-estradiol will attenuate stretching-induced cardiac dysfunction in an isolated heart model. The mechanisms could involve in blunting p38 MAPK and NF-κB signaling.