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.<p>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.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / Cardiovascular disease is the number one cause of death for Americans. New treatments are needed for serious conditions like atherosclerosis, as it can lead to stroke and heart attack. Many types of cells contribute to the progression of cardiovascular disease, including smooth muscle cells that comprise the middle layers of arteries. Inappropriate growth and migration of smooth muscle cells into the lumen of arteries has been implicated in vascular diseases. Death associated protein kinase (DAPK) is a protein that has been found to regulate the survival and migration of cancer cells, but has not been well characterized in vascular cells. The objective of this work was to determine the signaling pathways that DAPK regulates in smooth muscle cells. These studies have focused on smooth muscle cells isolated from human coronary arteries (HCASM cells). We have determined that HCASM cells depleted of DAPK exhibit more rapid migration, showing that DAPK negatively regulates migration of vascular cells. Results from a focused RT-PCR array identified matrix metalloproteinase 9 (MMP9) as a gene that is increased in cells depleted of DAPK. MMP9 is an important enzyme that degrades collagen, a component of the extracellular matrix through which smooth muscle cells migrate during atherosclerosis. We found that DAPK regulates phosphorylation of the NF-kappa B transcription factor p65 at serine 536, a modification previously found to correlate with increased nuclear levels and activity of p65. In DAPK-depleted HCASM cells, there was more phosphorylation of p65, which causes increased MMP9 promoter activity. Additional experiments were conducted using transgenic mice in which the DAPK gene has been deleted. By studying these mice, we have determined that under some circumstances DAPK augments maximal MMP9 levels in mouse carotid arteries which have been injured by ligation surgery via other signaling pathways. MMP9 has been previously implicated as a protein that promotes vascular diseases such as atherosclerosis. Our research in identifying DAPK as a regulator of MMP9 expression identifies a new target for treatment of vascular diseases like atherosclerosis.

GATA-4 est un facteur de transcription essentiel dans la formation du tube cardiaque primitif. Dans l'intestin, GATA-4 joue un rôle dans le développement et dans le maintien de l'identité jujéno-iléale. L'analyse par micropuce à ADN effectuée sur des cellules IEC-6 surexprimant GATA-4 suggère un rôle de GATA-4 dans la réponse inflammatoire et des études antérieures réalisées au laboratoire ont permis de confirmer l'implication de GATA-4 dans la modulation de la réponse inflammatoire C/EBPdépendante. L'objectif de mon projet était de déterminer si GATA-4 module la réponse inflammatoire par d'autres mécanismes que ceux déjà découverts au laboratoire et de générer des mutants de GATA-4 pour caractériser l'implication de certains domaines et sites spécifiques de GATA-4 dans la modulation de la réponse inflammatoire intestinale. Des cellules IEC-6 surexprimant GATA-4 ou des mutants de GATA-4 de façon stable, induites ou non à l'IL-1[béta] ont été utilisées comme modèle. L'activation des voies MAPK et Akt a été vérifiée par immunobuvardage de type Western. La liaison à l'ADN des facteurs de transcription AP-1, C/EBPs et NF-[kappa]B a été vérifiée par gel de rétention. L'expression de certains gènes de réponse inflammatoire a été vérifiée par RT-PCR. Finalement, des lignées cellulaires exprimant des mutants de GATA-4 ont été partiellement caractérisées. Mes résultats montrent qu'une surexpression de GATA-4 dans les IEC-6 traitées à l'IL-1[béta] réduit la phosphorylation de p42/p44 et d'Akt. Les facteurs de transcription AP-1, C/EBP et NF-[kappa]B sont davantage recrutés à l'ADN. La nature de leurs complexes semble être également affectée. Cette induction de liaison coïncide avec une baisse d'expression des gènes pro-inflammatoires CCL5 et iNOS. La caractérisation des mutants de GATA-4 suggère que le premier doigt de zinc (216-240) pourrait moduler la morphologie cellulaire et que la région basique (294-336) pourrait contribuer à la vitesse de prolifération augmentée des cellules IEC-6 surexprimant GATA-4. En somme, mes résultats suggèrent que GATA-4 pourrait participer à l'instauration d'une tolérance immunitaire prenant place lors de la différenciation entérocytaire.

Metastatic melanoma is an aggressive, incurable cancer with historically few therapeutic options. The discovery that 60% of melanomas harbor the oncogenic BRAF_V600E mutation, which constitutively activates the MAPK pathway, has provided a promising new therapeutic axis. Although MAPK pathway inhibitor therapy has shown striking clinical results in BRAF_V600-mutant melanoma, this approach faces three limitations. First, 10-20% of BRAF_V600-mutant melanomas never achieve meaningful response to MAPK pathway inhibitor therapy (intrinsic resistance). Second, among BRAF_V600-mutant melanomas initially responding to MAPK pathway inhibitor therapy, relapse is universal (acquired resistance). Third, approximately 40% of melanomas lack BRAF_V600 mutations and so are not currently candidates for MAPK pathway inhibitor therapy. We sought to address each of these problems: by characterizing the phenomenon of intrinsic MAPK pathway inhibitor resistance, by finding ways to perturb mechanisms of acquired MAPK pathway inhibitor resistance, and by identifying novel dependencies in melanoma outside of the MAPK pathway. Intriguingly, the NF-kappa B pathway emerged as a common theme across these investigations. In particular, we establish that MAPK pathway inhibitor sensitive and resistant melanomas display distinct transcriptional signatures. Unlike most BRAF_V600-mutant melanomas, which highly express the melanocytic lineage transcription factor MITF, MAPK pathway inhibitor resistant lines display low MITF expression but high levels of NF-kappa B signaling. These divergent transcriptional states, which arise in melanocytes from aberrant MAPK pathway activation by BRAF_V600E, remain plastic and mutually antagonistic in established melanomas. Together, these results characterize a dichotomy between MITF and NF-kappa B cellular states as a determinant of intrinsic sensitivity versus resistance to MAPK pathway inhibitors in BRAF_V600-mutant melanoma. In separate investigations, we have shown that, NFKB1 p105, a member of the NF-kappa B family, intimately regulates levels of COT, a known effector of resistance to MAPK pathway inhibitors. Moreover, we have used shRNA screening to nominate particular nodes within the NF-kappa B pathway, including MYD88 and IRF3, as candidate melanoma lineage-specific dependencies. Cumulatively, although these studies use diverse approaches to investigate the limitations of MAPK pathway inhibitor therapy in melanoma, they converge in nominating the NF-kappa B pathway as a previously underappreciated feature of melanoma biology and suggest the relevance of this pathway for future investigation.

In this study, we sought to define an underlying molecular mechanism of how inflammation induces cancer initiation. Cancer-associated inflammation is marked by the presence of inflammatory cells and mediators including cytokines, chemokines, and reactive oxygen species. There is a growing body of evidence establishing the link between chronic inflammation and cancer. Twenty percent of cancers have been linked to chronic infections. For instance, bacterial and viral infections induce inflammation which is a known risk factor for cancer. During inflammation, Ml macrophages' production of pro-inflammatory cytokines and reactive oxygen species (ROS) drives their function as anti-microbial. Likewise, the transcription factor nuclear factor kappa B (NF-KB) is known to induce a variety of stimulators, including ROS, to contribute to the inflammatory process. Therefore, we sought to explore the relationship between Ml macrophages and NF-KB, suggesting that Ml macrophage mediates cancer initiation via a NF-KB-dependent pathway, which collectively contributes to a metastatic phenotype.

Les protéines receptor-interacting protein kinase 1 (RIP1) et RIP3 ont été identifiées comme intervenant dans la régulation de la mort cellulaire apoptotique ou nécroptotique mais également dans la survie cellulaire. Ces deux protéines possèdent un domaine sérine/thréonine kinase, un domaine d’interaction spécifique RHIM (RIP homotypic interacting motif) et diffèrent dans leur domaine C-terminal car seule RIP1 possède un domaine de mort. Ces protéines font partie d’un ensemble de protéines régulatrices nommé ripoptosome. Des études ont montré une altération du ripoptosome dans les leucémies lymphoïdes chroniques (LLC) et les leucémies aigües lymphoïdes (LAL). Nous nous sommes intéressés aux leucémies aigües myéloïdes (LAM). L’analyse de l’expression des protéines RIP1 et RIP3 a été réalisée dans des blastes triées CD34+ de patients atteints de LAM ou dans des cellules CD34+ de donneurs sains en Q-RT-PCR. Les premières analyses montrent que RIP3 est significativement sous-exprimée chez les patients atteints de LAM en comparaison avec les cellules CD34+ issues de donneurs sains. Aucune différence n’a été mise en évidence pour l’expression de RIP1 dans les deux types de cellules CD34+. Afin de comprendre l’implication de l'extinction de RIP3 dans les LAM, nous avons étudié sa réexpression dans une lignée cellulaire leucémique murine (DA1-3b) où RIP3 n’est pas exprimée par métylation de son promoteur, au moyen d'un système d’expression conditionnelle (LacSwith II, IPTG). Après 10h d’induction de l’expression, on constate que la protéine RIP3 sauvage (RIP3-WT) induit une apoptose dans les cellules DA1-3b. Afin de déterminer l’implication des domaines de RIP3, nous avons utilisé une protéine mutante kinase Dead (RIP3-KD, activité kinase abolie) et une protéine mutante dans la séquence d’interaction spécifique avec RIP1 (RIP3-RHIM). L’analyse de la mortalité cellulaire en cytométrie en flux et en microscopie électronique montre que les protéines RIP3-WT et -KD induisent toutes les deux la mort apoptotique des cellules DA1-3b respectivement de 15% et de 50% après 10h d’expression. On constate donc que la protéine RIP3-KD induit une mort plus importante et plus précoce que la protéine sauvage. La protéine RIP3 mutée dans son domaine RHIM ne peut plus induire de mort cellulaire. Il semble donc que le domaine kinase de RIP3 jouerait un rôle régulateur dans la mort cellulaire induite par RIP3. L’utilisation du modèle de leucémie murine DA1-3b a permis de réaliser un étude in vivo de l’expression conditionnelle de RIP3-WT et -KD. Seule l'expression de RIP3-KD est capable de prolonger significativement la survie des souris.De plus, il a été démontré que RIP3 pouvait également induire la nécroptose dans les cellules lorsque l’apoptose ne peut aboutir, notament lorsque les caspases sont inhibées à l’aide d’un inhibiteur de pan-caspases le Z-VAD-FMK. Le traitement des cellules exprimant RIP3-WT par 50µM de Z-VAD-FMK induit une plus forte mortalité (45%) des cellules tandis que dans les cellules exprimant RIP3-KD, l’inhibiteur des caspases inhibe complètement le processus apoptotique et permet la survie des cellules (10%). Une étude en microscopie électronique a permis de déterminer que la présence de Z-VAD-FMK induit un switch de l’apoptose vers la nécroptose. Il semble donc que le domaine de kinase possède un rôle important dans la signalisation de la nécroptose car la protéine RIP3-KD n’est plus capable d’initier le switch entre l’apoptose et la nécroptose. Quelques données préliminaires semblent indiquer que les calpaïnes ainsi que la caspase 12 pourraient également être impliquées dans la balance apoptose/nécroptose. [...] / The receptor-interacting protein kinase 1 (RIP1) and 3 (RIP3) are key signaling molecules in the regulation of apoptotic cell death or in the execution of a specific instance of regulated necrosis, named necroptosis, as well as in cell survival processes. These proteins have in common a serine/threonine kinase domain and a specific interacting motif RHIM (RIP homotypic interacting motif), while they differ in their C-terminal domain, as only RIP1 is characterized by a death domain. They belong to a family of regulatory proteins forming a cell death-inducing platform, referred to as Ripoptosome. Previous studies showed that the ripoptosome was altered in chronic lymphoid leukemia (CLL) and acute lymphoid leukemia (ALL). We decided to focus our studies on acute myeloid leukemia (AML).Expression profile of RIP1 and RIP3 was established by Q-RT PCR on CD34+ sorted cells of AML patients or healthy donors. Our first results show that if RIP3 is significantly under expressed in CD34+ cells of AML patients compared to healthy donors, there was no difference in RIP1 expression pattern in both cell types.To further understand the functional relevance of RIP3 down-regulation in the leukemia, we used a murine leukemic cell line (DA1-3b) in which RIP3 promoter is methylated, inhibiting its expression.A conditional expression system in DA1-3b cells has been realized (LacSwith II). IPTG (Isopropyl-beta-D-thiogalactoside) treatment (1mM) allows expression of the proteins of interest in these cell lines. We noticed that, 10 hours after expression’s induction, the wild-type RIP3 protein (RIP3-WT) induces apotptosis in DA1-3b cells. In order to decipher the role of each RIP3 domains in this cell-death-induced phenomenon, several mutants were employed. We used a mutant protein with a non functional kinase domain, RIP3 Kinase Dead (KD) and a mutant unable to interact with RIP1, RIP1-RHIM. Cell death analysis, performed by flow cytometry and by electron microscopy, shows that both RIP3-WT and RIP3-KD induce apoptosis in DA1-3b cells (respectively 15% and 50%) after 10 hrs of expression. However, these results show that RIP3-KD induces a stronger and more rapid cell-death than the wild-type protein. In agrement with previous findings, we found that the protein mutated in the RHIM domain cannot engage a cell-death process. Our results thereby suggest that the RIP3 kinase domain palys a major regulatory role in cell-death.Taking advantage of a mouse model of leukemia, we also performed an in vivo study of conditional expression RIP3-WT and –KD. As a matter of fact, DA1-3b cells, obtained from C3H mice are able to induce a leukemia in these mice after transplantation. We have shown that the inducible expression of RIP3-KD in DA1-3b cells can increase significatively mice survival. Moreover, we have shown that, in certain conditions, RIP3 could also induce necroptosis when apoptosis is prevented by a pan-caspase inhibitor, Z-VAD-FMK. Treating RIP3-WT expressing cells with 50µM of Z-VAD-FMK induces a strong mortality (45%) while it inhibits totally the apoptotic process in RIP3-KD expressing cells, allowing cell survival (10%).Using electronic microscopy, we were able to demonstrate that the use of Z-VAD-FMK leads to a switch from apoptosis to necroptosis. Thus, as the kinase dead protein is not able to induce this switch, the kinase domain could play an important role in necroptotic signaling. Preliminary results suggest that calpains, as well as caspase 12, could also be involved in apoptosis/necroptosis balance. It has been shown that RIP1 and RIP3 could play a role in Nf-kB pathway regulation. Studying the effects of RIP3-KD expression on Nf-kB pathway, we were able to demonstrate that it led to a specific cleavage of Nf-kB p65 protein, forming two fragments of about 25 kDa and 40 kDa. [...]

A vasculopatia crônica do transplante constitui um dos principais obstáculos para o sucesso do transplante de órgãos a longo prazo. Caracteriza-se pela formação de uma camada neoíntima, que culmina no estreitamento da luz do vaso com consequente isquemia e falência do órgão transplantado. Embora diversos mecanismos imunológicos tenham sido descritos, a patogênese da vasculopatia crônica do transplante ainda não foi esclarecida e continua sem tratamento específico. Neste contexto, estratégias com alvos imunomodulatórios são de extrema importância. Dentre estas possíveis estratégias destaca-se a talidomida, uma droga que apresentada potentes propriedades anti-inflamatórias e imunomodulatórias e que recentemente voltou a ter indicações clínicas. Assim, os objetivos do presente estudo foram: 1) Padronizar o modelo experimental de vasculopatia crônica do transplante no modelo experimental de transplante de aorta, pois este modelo mimetiza as principais características observadas na parede do vaso durante o processo de rejeição; 2) Analisar o efeito da talidomida sobre as alterações morfológicas vasculares (histologia para Verhoeff), componentes celulares (expressão de a-actina, atividade proliferativa, número de macrófagos e linfócitos T, através de imuno-histoquímica), além da participação da apoptose (técnica de TUNEL); 3) Avaliar o potencial efeito anti-inflamatório e imunomodulador da talidomida neste modelo, através da análise da expressão de mediadores da resposta imune (TNF-a, IL-1b, IL-6, IL-2, INF-g, IL-4 e IL-10), bem como a atividade do NFkB e o envolvimento das subunidades p50 e p65 no enxerto vascular. Foram utilizados ratos machos das linhagens isogênicas específicas, Fisher 344 e Lewis, distribuídos em 3 diferentes grupos (32 por grupo): ISO (transplante isogênico de aorta, Fisher para Fisher); ALO (transplante alogênico de aorta, Fisher para Lewis); ALO+TALID (transplante alogênico de aorta tratado com talidomida (200mg/Kg/dia/gavagem)). Os animais foram acompanhados por um período de 30 dias e, em seguida, sacrificados. Os resultados obtidos demonstraram que a padronização do modelo experimental de transplante de aorta foi viabilizada dentro de uma sequência metodológica extremamente precisa e reprodutível. Como esperado, os animais do grupo ISO não apresentaram sinais de rejeição no enxerto vascular, mantendo íntegra a arquitetura das camadas do vaso. Por outro lado, animais do grupo ALO desenvolveram vasculopatia crônica do transplante caracterizada pelo espessamento das camadas íntima e adventícia. Na camada média evidenciou-se diminuição das VSMC e acentuado rompimento das fibras elásticas. Além disso, os animais do grupo ALO apresentaram aumento da expressão de a-actina acompanhada de intensa atividade proliferativa celular, particularmente nas camadas neoíntima e adventícia, provavelmente pela migração das VSMC para a camada íntima. A análise por IH revelou intenso infiltrado de macrófagos, linfócitos T, além de apoptose presente em todas as camadas do aloenxerto. A expressão gênica e a concentração tecidual dos mediadores da resposta imune TNF-a, IL-1b, IL-6, IL-2, INF-g e IL-10 também foram significativamente aumentadas no grupo ALO. Esses achados associaram-se com a ativação do NFkB, bem como o envolvimento dos dímeros p50 e p65. O tratamento com talidomida promoveu um efeito vasculoprotetor através da redução da camada neoíntima e da inflamação local. Além disso, induziu diminuição da expressão gênica e da concentração tecidual dos mediadores TNF-a, IL-1b, IL-6, IL-2, INF-g, bem como aumento da IL-4 e IL-10. A talidomida também diminuiu a ativação do NFkB. Conclusão: os resultados do presente estudo indicam que o modelo experimental de vasculopatia crônica do transplante constitui um modelo adequado para estudar as alterações nas camadas do vaso durante o processo de rejeição ao aloenxerto. A talidomida exerceu um efeito vasculoprotetor, atenuando a formação da neoíntima e do processo inflamatório local, bem como dos mediadores da resposta imune, provavelmente devido as suas propriedades anti-inflamatórias e imunomodulatórias / Chronic transplant vasculopathy is a major obstacle for the long-term success of organ transplantation. It is characterized by the formation of neointimal layer that culminate in the narrowing of the vessel lumen with consequent ischemia and failure of the transplanted organ. Although several immunological mechanisms have been described, the pathogenesis of chronic transplant vasculopathy remains unclear and continues without specific treatment. In this context, immunomodulatory strategies are extremely important. Among these possible strategies stands out thalidomide, a drug that display a potent antiinflammatory and immunomodulatory properties and recently have returned to clinical indications. The aims of this study were: 1) To standardize the experimental model of chronic transplant vasculopathy, induced in an experimental model of aortic transplantation, because this model mimics the key features observed in the vessel wall during the rejection process; 2) To analyze the effects of thalidomide on vascular morphological (Verhoeff staining), cellular components (a-actin expression, proliferative activity, number of macrophages and T lymphocytes by immunohistochemistry), and the participation of apoptosis (TUNEL assay); 3) To evaluate the antiinflammatory and immunomodulatory potential effects of thalidomide in this model by analyzing the mediator expression of the immune response (TNF-a, IL-1b, IL-6, IL-2, INF-g, IL-4 e IL-10), as well as the NFkB activity and involvement of p50 and p65 subunits in the vascular graft. We used male rats from the specific inbred strains, Fisher 344 and Lewis, divided into 3 different groups (32 per group). ISO (isogeneic aortic transplantation, Fisher to Fisher); ALO (allogeneic aortic transplantation, Fisher to Lewis); ALO+THALID (allogeneic aortic transplantation treated with thalidomide (200mg/Kg/day/gavage)). The animals were followed during a period of 30 days and then sacrificed. The results showed that the standardizing of the experimental model of aortic transplantation was possible within a sequence highly accurate and reproducible methodology. As expected, the animals of the ISO showed no sign of graft rejection keeping the entire layered architecture of the vessel. On the other hand, the ALO group developed chronic transplant vasculopathy characterized by thickness of the intima and adventitia. Furthermore, ALO group animals showed increase of a-actin expression accompanied by an intense cellular proliferative activity, particularly in the neointima and adventitia layers, probably due to VSMC migration to the intima. Analysis by IH revealed intense infiltration of macrophages and T lymphocytes, as well as apoptosis in all layers of the allograft. The gene expression and tissue concentrations of the mediators of immune response TNF-a, IL-1b, IL-6, IL-2, INF-g, and IL-10 were also significantly higher in the ALO group. These findings were associated with the NFkB activation, as well as the involvement of p50 and p65 dimers. Treatment with thalidomide promoted a vascular protective effect by reducing the neointimal formation and local inflammation. Moreover, induced a decrease in the gene expression and tissue concentration of the inflammatory mediators TNF-a, IL-1b, IL-6, IL-2, INF-g, as well as increase of IL-4 and IL-10. Thalidomide also decreased the NFkB activation. Conclusion: the results of this study indicate that experimental model of chronic transplant vasculopathy is an appropriate model to study changes in the vessel layers during the process of allograft rejection. Thalidomide promoted a vascular protective effect, attenuating neointimal formation and local inflammation, as well as mediators of immune response, probably due to its anti-inflammatory and immunomodulatory properties

While prompt activation of NF-κB is essential for optimal immune response, it is equally important to terminate the response to avoid tissue damage and perhaps even death resulting from organ failure. This thesis describes two inhibitors of NF-κB, A20 and IκBβ. A20 is an essential inhibitor of NF-κB mediated inflammation as mice lacking A20 die from multi-organ inflammation and cachexia. Multiple biochemical approaches have suggested that A20 functions as a deubiquitinase by disassembling K63-linked regulatory ubiquitin chains from upstream adapter molecules like RIP1. To determine the contribution of the deubiquitinase role of A20 in downregulating NF-κB, we generated and characterized a knock-in mouse lacking the deubiquitinase activity of A20. However, we find that these mice display normal NF-κB activation and show no signs of inflammation. Our results suggest that the deubiquitinase activity of A20 is dispensable for downregulating NF-κB. The second part of this thesis unravels a new biological pathway mediated by IκBβ. Unlike IκBα, which functions solely as an inhibitor of NF-κB, IκBβ can both inhibit and activate NF-κB depending on the physiological context. We hypothesized that this may be because IκBβ (unlike IκBα ) exists in two forms, a constitutively phosphorylated form and an unphosphorylated form. Prior work from our group has demonstrated that hypophosphorylated IκBβ complexes with p65:cRel and mediates the expression of certain inflammatory genes like TNFα . We report here that Glycogen Synthase Kinase 3β (GSK-3β ) interacts with and phosphorylates IκBβ at Serine-346. This phosphorylation masks the NLS of p65 in the phospho-IκBβ:p65:cRel complex, thereby sequestering the complex in the cytoplasm and mediating the anti-inflammatory role of IκBβ. We discovered a peptide that can inhibit this phosphorylation by abrogating the interaction between GSK-3β and IκBβ. Mice succumb to a sublethal dose of LPS when injected with this peptide because of increased production of TNFα (but not IL-6); thereby demonstrating the inflammatory role of unphosphorylated IκBβ in upregulating specific genes like TNFα. We propose a signaling model by which phosphorylation by GSK-3β can regulate the functions of IκBβ in response to LPS.

The nuclear factor-kB (NF-kB) signaling cascade is comprised of two branches, the canonical and alternative NF-kB pathways. Signaling through the alternative NF-kB pathway culminates in the activation of the downstream transcription factor subunits, RELB and NF-kB2. The biological roles of RELB and NF-kB2 within the B cell lineage have been obscured in constitutional knockout mice by the diverse functions of these subunits in non-B cell types. To overcome these limitations, conditional alleles were generated to investigate the roles of RELB and NF-kB2 in B cell development. These alleles allowed the identification of complex functional requirements for RELB and/or NF-kB2 in naïve B cells, germinal center (GC) B cells and plasma cells (PCs). These functional requirements may have implications for B cell malignancies that display mutations that constitutively activate the alternative NF-kB pathway. A large body of work has demonstrated that B cell activating factor (BAFF) signaling is critical for the maintenance of mature B cells. However, the contribution of the alternative NF-kB subunits that are activated downstream of BAFF remained unclear, especially in regards to their specific target genes. We have identified critical, B cell-intrinsic roles for RELB and NF-kB2 in the maintenance of mature B cells. In response to BAFF, these subunits were found to control the expression of anti-apoptotic genes, genes that ensure correct positioning within the B cell niche, and genes involved in promoting B–T cell interactions that allow effective antigen-mediated activation. During the GC B cell reaction, light zone (LZ) B cells undergo affinity-based selection mediated by T follicular helper (Tfh) cells. A subset of LZ B cells show activation of the NF-kB signaling cascade, suggesting a critical role for NF-kB in the selection of high-affinity GC B cells. We here report that GC B cell development occurred normally in mice with conditional deletion of either relb (RELB) or nfkb2 (NF-kB2) in GC B cells. In contrast, the simultaneous ablation of both subunits caused rapid involution of established GCs, similar to what has been observed for ablation of the canonical NF-kB transcription factor subunit c-REL. Intriguingly, RNA-sequencing analysis of relb/nfkb2-deleted GC B cells revealed no overlap between the genes controlled by RELB/p52 and c-REL within GC B cells. This suggests that signaling through the separate NF-kB pathways in GC B cells results in the expression of different biological programs that are independently required for the maintenance of the GC reaction. In addition, we observed that human PCs and PC precursors within the LZ showed high protein levels of NF-kB2 compared to surrounding lymphocytes, suggesting a biological role for this subunit in PCs. Indeed, ablation of nfkb2 alone in GC B cells led to a dramatic decrease in antigen-specific serum IgG1 and antigen-specific IgG1-secreting cells. Interestingly however, the mice developed normal frequencies of PCs, suggesting a role for NF-kB2 in PC physiology rather than differentiation.

Introdução: O Diabetes Mellitus é doença que apresenta elevada incidência e prevalência na população em diversas partes do mundo. Estudos experimentais sugerem que o estresse oxidativo esteja envolvido na patogênese e na progressão dela. Objetivo: Investigar os efeitos da utilização exógena do antioxidante orgoteína (SOD Cu/Zn) sobre o estresse oxidativo, ativação do fator de transcrição nuclear kappa B (NFκB) e expressão da óxido nítrico sintase induzível (iNOS) hepática no modelo experimental de diabetes mellitus (DM). Métodos e Resultados: Foram utilizados 28 ratos machos Wistar, divididos em quatro grupos: controle (CO), controle tratados com orgoteína (CO+ORG), diabéticos (DM) e diabéticos tratados com orgoteína (DM+ORG). A dose utilizada da ORG (orgoteína ®) foi de 13 mg/Kg de peso corporal durante os sete últimos dias de experimento através de injeção subcutânea. O DM foi induzido por injeção de estreptozotocina (i.p.,70 mg/kg) e, após sessenta dias, foi avaliada a lipoperoxidação hepática através das substâncias que reagem ao ácido tiobarbitúrico (TBARS- nmoles/mg de proteína), a atividade das enzimas antioxidantes catalase (CAT-pmoles/mg de proteína), superóxido dismutase (SOD-U/mg de proteína) e glutationa peroxidase (GPx- nmoles/mg de proteína). Também foi realizada a medida de nitritos e nitratos (mM de NO2 e NO3). As expressões da p65 do NFκB e da iNOS foram avaliadas pela técnica de Western blot e quantificadas por densitometria em relação à percentagem do controle. Resultados: Houve aumento na lipoperoxidação hepática no grupo DM e redução significativa no grupo DM+ORG (0,36±0,15 vs 0,18±0,04; p<0,01). A atividade da enzima CAT apresentou aumento no grupo DM e redução significativa no grupo DM+ORG (p<0,001), enquanto a atividade da SOD e da GPx apresentaram redução no DM e aumento no grupo DM+ORG, respectivamente (p<0,001 e p<0,05). A medida de nitritos e nitratos apresentou redução no DM e aumento no grupo DM+ORG (1,47±0,61 vs 2,29±0,20; p<0,05) enquanto a expressão da iNOS foi 32% maior no grupo DM em relação ao grupo CO e 53% maior no grupo DM+ORG, quando comparado com o grupo DM (p<0,01). A p65 foi 37% mais expressa no DM (p<0,05) e sem diferença estatística, quando comparado o grupo DM com o grupo DM+ORG. Conclusão: O tratamento com orgoteína reduziu o estresse oxidativo hepático em animais diabéticos, mesmo não alterando o fator de transcrição nuclear NFкB. A orgoteína também aumentou a bioatividade do NO, provavelmente, devido ao aumento da atividade, dismutando o radical superóxido, e devido ao aumento da expressão da iNOS. / Aim: To investigate the effects of exogenous antioxidant orgoteína on oxidative stress, activation of nuclear transcription factor kappa B (NFκB), and hepatic expression of inducible nitric oxide synthase (iNOS) in the experimental model of diabetes mellitus (DM). Methods: 28 male Wistar rats divided in four groups were used: control (CO), controls treated with orgoteína (CO+ORG), diabetics (DM), and diabetics treated with orgoteína (DM+ORG). Orgoteína (orgotein®, 13 mg/Kg body weight was administered subcutaneously for the last 7 days of the trial. DM was induced by a single streptozotocin injection (i.p.,70 mg/kg), and 60 days later, we evaluated liver lipoperoxidation through thiobarbituric acid reactive substances (TBARS- nmoles/mg of protein) and activity of antioxidant enzymes catalase (CAT-pmoles/mg of preotein), superoxide dismutase (SODU/ mg of protein) and glutathione peroxidase (GPx- nmoles/mg of protein). Nitrites e nitrates (mM NO2 and NO3) were also measured, and p65 of NFκB and iNOS expressions were evaluated by Western blot and quantified by densitometry as compared to control percentage. Results: Liver peroxidation was increased in the DM group and significantly decreased in the DM+ORG group (0.36±0.15 vs 0.18±0.04; p<0.01). CAT activity was increased in the DM group and significantly decreased in the DM+ORG group (p<0.001), whereas SOD and GPx activity were decreased in the DM group and increased in the DM+ORG group, respectively (p<0.001 and p<0.05). Nitrite and nitrate measures were reduced in the DM and increased in the DM+ORG group (1.47±0.61 vs 2.29±0.20; p<0.05), while iNOS expression in the DM group was 32% greater than in the CO and 53% greater in the DM+ORG group than in the DM group (p<0.01). P65 expression was 37% higher in the DM (p<0.05), and there was no significant difference between the DM and DM+ORG groups. Conclusion: Orgoteína treatment reduced liver oxidative stress in diabetic animals, even though it did not change NFкB. Orgoteína also increased NO bioactivity, probably by the increased dismutation of the superoxide radical and by increase in iNOS expression.