京都大学 / 新制・課程博士 / 博士(医学) / 甲第24503号 / 医博第4945号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 椛島 健治, 教授 後藤 慎平 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Le système immunitaire inné est un mécanisme de défense commun à tous les métazoaires. Chez l’Homme comme chez la drosophile, son activation peut être délétère lorsqu’elle est incontrôlée. L’étude des mécanismes qui sous-Tendent cet équilibre entre l’activation ou non de la réponse immunitaire innée est à la base de mes travaux de thèse. En utilisant le modèle Drosophila melanogaster, j’ai caractérisé la protéine Big-Bang comme un acteur important de la balance immunitaire intestinale. Mes résultats démontrent que Big-Bang est un constituant des jonctions obturantes de l’épithélium intestinal. Son absence provoque une rupture de tolérance immunitaire envers la flore bactérienne endogène et d’autre part une sensibilité accrue aux pathogènes invasifs. Mes travaux de thèse ont également permis de caractériser Akirine, une protéine nucléaire qui agit au niveau des facteurs NF-ΚB de la drosophile à l’Homme. Mes résultats démontrent qu’Akirine est un sélecteur qui agit de concert avec le complexe de remodelage de la chromatine SWI/SNF et NF-ΚB pour transcrire un sous-Ensemble de gènes pro-Inflammatoires. / The innate immune response is required by all metazoan to defend themselves against microorganisms. When abnormally activated however, innate immune responses cause deleterious chronic inflammation. The study of the fragile equilibrium between immune responses and tolerance has fundamentally shaped the projects of my PhD work.First, using Drosophila melangoaster as a model, I characterized Big-Bang as a major player of the immune balance in the gut. I could show that Big-Bang is a bona fide component of midgut epithelium septate junctions. Consequently, big-Bang deficient flies have an impaired tolerance against commensal microorganisms and are susceptible to invasive gut pathogens, ultimately leading to a premature death of flies.I focused the second part of my PhD work on the characterization of Akirin, a nuclear protein required for the activation of NF-ΚB response from Drosophila to humans. My results showed that Akirin is a selector molecule, acting together with NF-ΚB and the SWI/SNF chromatin-Remodeling complex to sustain the transcription of a subset of pro-Inflammatory genes.

A l’instar du lymphome de Burkitt (LB) avec la translocation de MYC, les lymphomes diffus à grandes cellules B (DLBCL) par d'autres mécanismes (mutation, amplification, dérégulation du promoteur) sont associés à une dérégulation de c-Myc, facteur de transcription maitre de la prolifération. Les DLBCL sont classés en deux sous-groupes: « centre germinatif » (GCB) et « cellule B activée » (ABC) avec activation constitutive de NF-κB. Cette activation constitutive de NF-κB peut être le résultat d'altérations génétiques (MYD88, A20, TRAF2 et TRAF5) ou de l'activation du BCR ou CD40. Ces caractéristiques soulèvent la question de la synergie d’action entre NF-κB et c-Myc dans les ABC-DLBCL. Nous avons analysé l’effet d'une activation continue de c-Myc dans un contexte de sur-activation de NF-κB par plusieurs inducteurs. Nos résultats montrent que la surexpression de c-Myc dans un contexte d'induction de NF-κB, i) par le programme EBV latence III, apporte un avantage sélectif à ces cellules (expression génique en faveur d'un métabolisme élevé, prolifération intense et protection contre apoptose), ii) par le TLR9 (modèle in vivo et in vitro), augmente la survie et la prolifération des lymphocytes B des souris λc-Myc (augmentation des cellules B activées, splénomégalie, augmentation de la prolifération des lymphocytes B, modification du microenvironnement tumoral), et iii) par CD40, induit une lymphomagenèse B très agressive dans les souris doubles transgéniques CD40/Myc, les tumeurs ont un phénotype proche des ABC-DLBCL. Ces résultats suggèrent que c-Myc est un événement co-transformant dans les lymphomes agressifs avec un phénotype activé par NF-κB, tel que les ABC-DLBCL. / Not only Burkitt lymphoma (BL) with the translocation of MYC, but also diffuse large B-cell lymphoma (DLBCL) by other mechanisms (mutation, amplification, promoter dysregulation…) are associated with dysregulation of c-Myc, the master transcription factor for proliferation. DLBCL’s are classified in two subgroups: “Germinal center B-cell” (GCB) without and “activated B-cell” (ABC) with constitutive NF-κB activation. This constitutive activation of NF-κB can be the result of genetic alterations (MYD88, A20, TRAF2, and TRAF5) or the activation of B-cell receptor or CD40. These features raise the question of the synergy of action between NF-κB and c-Myc in ABC-DLBCL. We analyzed the effect of a continuous activation of c-Myc in a context of over-activation of NF-κB by several inductors. Our results show that overexpression of c-Myc in the context of induction of NF-κB, i) by EBV latency III program, provides a selective advantage to those cells (gene expression in favor of a high metabolism, intense proliferation and protection against apoptosis), ii) by TLR9 (in vivo and in vitro model) increases the survival and proliferation of B lymphocytes of λc-Myc mice (increase of activated B cells, splenomegaly, increased B cells proliferation, modification of tumor microenvironment), and iii) by CD40, induces a very aggressive B lymphomagenesis in CD40/Myc double transgenic mice, the tumors have a phenotype close to ABC-DLBCL. These results suggest that c-Myc is an NF-κB co-transforming event in aggressive lymphomas with an activated phenotype by NF-κB, such as ABC-DLBCL.

BACKGROUND:Marek's Disease (MD) is a hyperproliferative, lymphomatous, neoplastic disease of chickens caused by the oncogenic Gallid herpesvirus type 2 (GaHV-2 / MDV). Like several human lymphomas the neoplastic MD lymphoma cells overexpress the CD30 antigen (CD30hi) and are in minority, while the non-neoplastic cells (CD30lo) form the majority of population. MD is a unique natural in-vivo model of human CD30hi lymphomas with both natural CD30hi lymphomagenesis and spontaneous regression. The exact mechanism of neoplastic transformation from CD30lo expressing phenotype to CD30hi expressing neoplastic phenotype is unknown. Here, using microarray, proteomics and Systems Biology modeling / we compare the global gene expression of CD30lo and CD30hi cells to identify key pathways of neoplastic transformation. We propose and test a specific mechanism of neoplastic transformation, and genetic resistance, involving the MDV oncogene Meq, host gene products of the Nuclear Factor Kappa B (NF-kappaB) family and CD30 / we also identify a novel Meq protein interactome.RESULTS:Our results show that a) CD30lo lymphocytes are pre-neoplastic precursors and not merely reactive lymphocytes / b) multiple transformation mechanisms exist and are potentially controlled by Meq / c) Meq can drive a feed-forward cycle that induces CD30 transcription, increases CD30 signaling which activates NF-kappaB, and, in turn, increases Meq transcription / d) Meq transcriptional repression or activation of the CD30 promoter generally correlates with polymorphisms in the CD30 promoter distinguishing MD-lymphoma resistant and susceptible chicken genotypes e) MDV oncoprotein Meq interacts with proteins involved in physiological processes central to lymphomagenesis.CONCLUSIONS:In the context of the MD lymphoma microenvironment (and potentially in other CD30hi lymphomas as well), our results show that the neoplastic transformation is a continuum and the non-neoplastic cells are actually pre-neoplastic precursor cells and not merely immune bystanders. We also show that NF-kappaB is a central player in MDV induced neoplastic transformation of CD30-expressing lymphocytes in vivo. Our results provide insights into molecular mechanisms of neoplastic transformation in MD specifically and also herpesvirus induced lymphoma in general.

The v-rel oncogene is the most efficiently transforming member of the Rel/NF-κB family of transcription factors. Identification of genes or signal transduction pathways that contribute to v-Rel transformation provide insight into the mechanisms of tumorigenesis by Rel/NF-κB proteins. In these studies, the contribution of TGF-β/Smad signaling to v-Rel transformation was assessed. TGF-β/Smad signaling regulates several cellular processes, including growth, differentiation, and apoptosis and has been implicated in a number of different cancers. Using microarray technology and Northern blot analysis, key components of the TGF-β/Smad pathway (tgf-β2 and tgf-β3 ligands, TGF-β type II receptor, and receptor-activated smad3) were identified with upregulated mRNA expression in v-Rel-transformed fibroblasts and lymphoid cells relative to control cells. A corresponding change in their protein levels was also observed. Further analysis revealed elevated levels of the phosphorylated, active form of Smad3, which correlated with its increased DNA-binding activity in v-Rel transformed cells. In contrast, the overexpression of c-Rel resulted in little to no alteration in the RNA and protein expression of members of the TGF-β/Smad pathway. Further studies demonstrated that elevated TGF-β/Smad signaling is required for the transforming ability of v-Rel. Blocking TGF-β signaling with a kinase inhibitor of TGF-β type I receptor inhibited the activation of Smad3 and dramatically reduced the ability of v-Rel transformed cells to form colonies in soft agar. Overexpression of a constitutively active form of Smad3 in the inhibitor-treated cells restored their ability to form colonies in soft agar close to the levels seen in untreated cells. Additional experiments with dominant negative Smad3 also revealed its ability to hinder the oncogenic potential of v-Rel. In complementary experiments, a stimulatory effect on v-Rel transformation was observed with cells treated with recombinant TGF-β2 ligand or overexpressed with wild-type Smad3. Taken together, these studies demonstrate that TGF-β signaling is crucial for the transformation potential of v-Rel and is primarily mediated by Smad3 activity. / text

The risk of injury, inflammation, and oxidative stress increases in skeletal muscle with aging. It has been postulated that pro-oxidant signaling, including upregulation of inducible nitric oxide synthase (iNOS) contributes to inflammation, pathology, and aging in the brain, liver and heart. Exercise training reduces the risk of injury and inflammation. The purpose of this study was: 1) to identify the mechanisms that upregulate iNOS, pro-oxidant and pro-inflammatory signaling in skeletal muscle, and 2) to identify the mechanisms by which exercise training reduces pro-oxidant signaling. Protein levels and activity of iNOS were measured in 4 groups of male Fischer-344 rats (5 mo and 24 mo, n=10/group), old-control (OC), old-trained (OT), young-control (YC), and young-trained (YT). Exercise training protocol was 60 min at 15 m/min at 15° incline for 5 d/wk for 12 wk. Both iNOS protein expression and activity were significantly higher in OC compared to YC, but exercise training reversed the elevation of iNOS levels lower than OC in tibialis anterior. Surprisingly, NF-κB DNA binding activity was significantly lower in OC than YC, while increased with exercise training in white and red gastrocnemius in both OT and YT. In contrast, protein expression of p65, a regulatory subunit of NF-κB was significantly greater in OC than YC, while exercise training significantly reduced p65 in OT compared to OC from the white gastrocnemius. These data indicate that regulation of NF-κB activity with aging is post-translational and alterations in iNOS expression may result from alternative NF-κB pathways. As decreased NF-κB activity with aging could result in downstream increase in pro-apoptotic signaling, we tested follow-up hypotheses that aging would increase pro-apoptotic regulator Bax and decrease the anti-apoptotic regulator Bcl-2. Bax increased while Bcl-2 decreased in OC in white gastrocnemius when compared to YC. In contrast, exercise training resulted in a dramatic upregulation of Bcl-2 and downregulation of Bax protein expression in OT when compared to OC. These novel results indicate that alterations in pro-inflammatory and pro-apoptotic signaling occur in skeletal muscle during the aging process. Importantly, our findings strongly support the hypothesis that exercise training reverses age-induced changes in pro-inflammatory and pro-apoptotic signaling.

Les facteurs de transcription de la famille Rel/NF-κB régulent l’expression d’un grand nombre de gènes impliqués dans les réponses immunitaires et inflammatoires ainsi que dans la régulation de la prolifération et de la survie cellulaire. Le caractère transitoire de l’activation de NF-κB est donc crucial pour poterger les cellules de l’autoxicité due à une trop forte expression des gènes cibles de ce facteur de transcription. Dans le cadre de notre thèse de doctorat, nous avons étudié les mécanismes moléculaires régulant la cinétique d’activation de NF-κB, en accordant une attention toute particulière au complexe kinase IKK, qui semble être le regulateur clef de l’activation de NF-κB. Nos résultats suggèrent que p300 pourrait réguler la durée d’activation des IKKs d’une part par acétylation directe, et d’autre part, indépendamment de son activité HAT, en stabilisant les IKKs et donc en prolongeant leur demie-vie et par conséquent leur activation. Certains virus utilisent la voie de signalisation NF-κB afin de promouvoir leur propre réplication. C’est le cas du virus HIV-1 (Human Immunodeficiency Virus type 1), qui contient dans son promoteur deux sites de liaison pour NF-κB. Notre laboratoire a précédemment montré que l’utilisation du TNFα en combinaison avec la TSA, active l’expression virale de manière synergique. L’administration combinée d’un activateur du facteur NF-κB et d’un inhibiteur de désacétylases pourrait, en présence d’une thérapie anti-HIV-1 efficace, être envisagée dans le but d’éliminer les cellules réservoirs infectées de manière latente. L’utilisation thérapeutique du TNFα ou de la TSA étant inenvisageable en raison de leur toxicité, nous avons étudié l’effet d’autres substances ayant un plus grand potentiel thérapeutique et nous avons apporté une preuve de principe du potentiel thérapeutique de la coadministration de plusieurs activateurs viraux (inhibiteurs de HDACs[HDACIs]+inducteurs de la voie NF-κB) pour réduire le pool des réservoirs cellulaires infectés de manière latente.

Recent genome-wide association studies identified FGFR2 as one of breast cancer susceptibility genes. FGFR2 expression was down-regulated in breast carcinomas when compared with paired normal epithelium. Stable retroviral transduction of FGFR2-IIIb and its alternatively spliced FGFR2-IIIc variants was achieved in breast cancer MDA-MB-231, T47D and near normal MCF-10A cells. Our findings revealed a direct reduction of breast cancer cell growth and motility, a significant arrest of transformed morphogenetic changes including the Epithelial to Mesenchymal transition (EMT), anchorage independent growth, and the formation of growth-arrested 3D acinar architectures, and suppressive actions on orthotopically xenografted epithelial neoplasms and surrounding tumor stroma. These tumor protective effects were concordant with physical interactions between the two FGFR2 isoforms and IKKβ. Consistent with these interactions we noted FGFR2 to inhibit NF-κB signaling, including decreased nuclear RelA/p65 NF-κB localization, down-regulation of a transfected NF-κB luciferase reporter, reduced production of NF-κB-dependent transcripts, Interleukin-6 and p-STAT3.

Recent genome-wide association studies identified FGFR2 as one of breast cancer susceptibility genes. FGFR2 expression was down-regulated in breast carcinomas when compared with paired normal epithelium. Stable retroviral transduction of FGFR2-IIIb and its alternatively spliced FGFR2-IIIc variants was achieved in breast cancer MDA-MB-231, T47D and near normal MCF-10A cells. Our findings revealed a direct reduction of breast cancer cell growth and motility, a significant arrest of transformed morphogenetic changes including the Epithelial to Mesenchymal transition (EMT), anchorage independent growth, and the formation of growth-arrested 3D acinar architectures, and suppressive actions on orthotopically xenografted epithelial neoplasms and surrounding tumor stroma. These tumor protective effects were concordant with physical interactions between the two FGFR2 isoforms and IKKβ. Consistent with these interactions we noted FGFR2 to inhibit NF-κB signaling, including decreased nuclear RelA/p65 NF-κB localization, down-regulation of a transfected NF-κB luciferase reporter, reduced production of NF-κB-dependent transcripts, Interleukin-6 and p-STAT3.

The transcription factor NF-κB interacts with many other co-regulator proteins that modulate its binding and transcriptional activity. One of these co-regulators, Pirin, is an iron-dependent metalloprotein that has been shown to enhance the DNA binding of NF-κB homodimers. Here, we characterize the interactions between Pirin and its known NF-κB binding partners and examined the role of Bcl-3, a protein that is required for Pirin’s interaction with p50. In addition, we use site-directed mutagenesis to alter conserved residues within Pirin’s metal binding environment and observed how it affected the DNA binding and conformation of the Pirin-NF-κB complex. These studies show that, while a similar enhancing effect on DNA binding is observed, the interactions of Pirin with different NF-κB members are distinct from each other and could possibly have different physiological purposes.