Comparison of NF-kB Regulation in Naive and Anergic Primary CD8+ T Lymphocytes

Due to the cytotoxic potential of CD8+ T cells, maintenance of CD8+ tolerance is critical. A major mechanism of peripheral tolerance in T lymphocytes is the induction of anergy, a refractory state caused by T lymphocyte activation in the absence of costimulation. Hallmarks of anergy are decreased IL-2 secretion and decreased proliferation. Here we used a T cell receptor transgenic mouse model to determine whether there are defects in the NF-κB signaling pathway in CD8+ T lymphocytes rendered anergic in vivo. In the anergic cell population, decreased NF-κB-mediated gene transcription and NF-κB p65 subunit DNA binding activity were observed. These changes were not due to inhibition of early NF-κB activation events, including IκBα degradation and NF-κB p65 subunit nuclear translocation, which occurred normally in anergic T lymphocytes. Nor were they related to defective phosphorylation of p65 at Ser536 in the cytoplasm or Ser276 in the nucleus, as p65 was phosphorylated at these residues in both naïve and anergic T lymphocytes with similar kinetics. However, the anergic CD8+ lymphocytes failed both to phosphorylate the NF-κB p65 subunit at Ser311 an event implicated in the recruitment of histone acetyl-transferase molecules such as CBP and p300, and to acetylate p65 at Lys310. Both of these posttranslational modifications have been shown to be critical for the positive regulation of NF-κB transcriptional activity. Thus, our results suggest that defects in key phosphorylation and acetylation events in p65 underlie defective NF-κB transactivation capacity and resultant lack of T cell function observed in anergic CD8+ T lymphocytes. Taken together these data provide a novel mechanistic explanation of how NF-κB p65 subunit is regulated in anergic CD8+T lymphocytes leading to defective NF-κB transcriptional activity and suggest that recruitment of CBP/p300 and p65 DNA binding in vivo is abrogated in anergic T lymphocytes

Understanding two inhibitors of NF-κB: A20 and IκBβ

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

Regulation of IκBα Function and NF-κB Signaling: AEBP1 Is a Novel Proinflammatory Mediator in Macrophages

NF-κB comprises a family of transcription factors that are critically involved in various inflammatory processes. In this paper, the role of NF-κB in inflammation and atherosclerosis and the regulation of the NF-κB signaling pathway are summarized. The structure, function, and regulation of the NF-κB inhibitors, IκBα and IκBβ, are reviewed. The regulation of NF-κB activity by glucocorticoid receptor (GR) signaling and IκBα sumoylation is also discussed. This paper focuses on the recently reported regulatory function that adipocyte enhancer-binding protein 1 (AEBP1) exerts on NF-κB transcriptional activity in macrophages, in which AEBP1 manifests itself as a potent modulator of NF-κB via physical interaction with IκBα and a critical mediator of inflammation. Finally, we summarize the regulatory roles that recently identified IκBα-interacting proteins play in NF-κB signaling. Based on its proinflammatory roles in macrophages, AEBP1 is anticipated to serve as a therapeutic target towards the treatment of various inflammatory conditions and disorders

Pathogenetics mechanisms in celiac disease

La tesi di Dottorato, svolta presso il Dipartimento di Farmacologia Sperimentale, redatta in lingua inglese ed intitolata “PATHOGENETICS MECHANISMS IN CELIAC DISEASE” ha riguardato lo studio dei meccanismi molecolari coinvolti nella patogenesi della Celiachia. I risultati della ricerca, condotta nel primo anno, ci hanno permesso di dimostrare che il fattore di trascrizione nuclear factor-B (NF-B) è attivato nella mucosa intestinale di pazienti celiaci indicando un ruolo per IB- nel regolare la persistente attivazione di NF-B in questa patologia. Le cellule epiteliali delle cripte e le cellule mononucleate della lamina propria di pazienti celiaci risultavano positive per p65 rispetto ai pazienti celiaci in remissione e di controllo. Inoltre, l'attivazione di NF-B era correlata con l'espressione della monossido d’azoto sintasi inducibile (iNOS) e della cicloossigenasi-2 (COX-2), enzimi che catalizzano la sintesi di monossido d’azoto (NO) e prostaglandine pro-infiammatorie. Queste osservazioni potrebbero essere di una certa rilevanza clinica poichè una sostenuta attivazione di NF-B nella mucosa intestinale di pazienti celiaci conduce ad una prolungata induzione dell'espressione di geni pro-infiammatori perpetuando in tal modo il processo infiammatorio cronico. I risultati della ricerca, condotta nel secondo e terzo anno, ci hanno permesso di approfondire alcuni meccanismi dell’interazione diretta della gliadina (un peptide del glutine) con i macrofagi murini RAW 264.7 stimolati con IFN-. In particolare, la gliadina in associazione con IFN- incrementava la produzione di NO, l’espressione della iNOS, l’attività di legame dei fattori di trascrizione NF-B, IRF-1 (interferon regulatory factor-1) e STAT-1 (signal transducer and activator of transcription-1 al DNA rispetto all’IFN- da solo. Questi effetti erano inibiti significativamente da pirrolidina ditiocarbammato, genisteina e tirfostina B42, rispettivamente, inibitori dell’attivazione di NF-B, IRF-1 e STAT-1. Approfondendo questi studi, è stato possibile accertare che la gliadina era in grado di aumentare i livelli di mRNA e l’attività del promotore del gene della iNOS nei macrofagi RAW 264.7 stimolati con IFN-per 1, 6 e 24 ore Questi effetti erano inibiti significativamente da genisteina e tirfostina B42 a 1 ora e da pirrolidina ditiocarbammato a 6 e 24 ore. E’ interessante osservare che le cinetiche di inibizione dell’espressione del gene della iNOS da parte di pirrolidina ditiocarbammato, genisteina e tirfostina B42 erano correlate con l’induzione dei livelli di mRNA dei fattori di trascrizione NF-B/p65, IRF-1 e STAT-1. Questi risultati suggeriscono che la gliadina può modulare l’espressione del gene della iNOS come co-segnale con l’IFN- attraverso IRF-1 e STAT-1 nelle fasi precoci e NF-B nelle fasi tardive dell’induzione. In conclusione, le nostre osservazioni possono contribuire ad una maggiore comprensione dei meccanismi molecolari della patogenesi della celiachia delineando nuove vie per il trattamento di questo disordine

Mechanisms of Legionella pneumophila-induced interleukin-8 expression in human lung epithelial cells

<p>Abstract</p> <p>Background</p> <p><it>Legionella pneumophila </it>is a facultative intracellular bacterium, capable of replicating within the phagosomes of macrophages and monocytes, but little is known about its interaction with human lung epithelial cells. We investigated the effect of <it>L. pneumophila </it>on the expression of interleukin-8 (IL-8) in human A549 alveolar and NCI-H292 tracheal epithelial cell lines.</p> <p>Results</p> <p>Infection of <it>L. pneumophila </it>strain, but not heat-killed strain, resulted in upregulation of IL-8. IL-8 mRNA expression was induced immediately after the infection and its signal became gradually stronger until 24 h after infection. On the other hand, IL-8 expression in A549 cells infected with <it>L. pneumophila </it>lacking a functional type IV secretion system was transient. The IL-8 expression was slightly induced at 16 h and increased at 24 h after infection with flagellin-deficient <it>Legionella</it>. Activation of the IL-8 promoter by <it>L. pneumophila </it>infection occurred through the action of nuclear factor-κB (NF-κB). Transfection of dominant negative mutants of NF-κB-inducing kinase, IκB kinase and IκB inhibited <it>L. pneumophila</it>-mediated activation of IL-8 promoter. Treatment with hsp90 inhibitor suppressed <it>L. pneumophila</it>-induced IL-8 mRNA due to deactivation of NF-κB.</p> <p>Conclusion</p> <p>Collectively, these results suggest that <it>L. pneumophila </it>induces activation of NF-κB through an intracellular signaling pathway that involves NF-κB-inducing kinase and IκB kinase, leading to IL-8 gene transcription, and that hsp90 acts as a crucial regulator in <it>L. pneumophila</it>-induced IL-8 expression, presumably contributing to immune response in <it>L. pneumophila</it>. The presence of flagellin and a type IV secretion system are critical for <it>Legionella </it>to induce IL-8 expression in lung epithelial cells.</p

Unlocking the NF-κB Conundrum: Embracing Complexity to Achieve Specificity.

Transcription factors of the nuclear factor κB (NF-κB) family are central coordinating regulators of the host defence responses to stress, injury and infection. Aberrant NF-κB activation also contributes to the pathogenesis of some of the most common current threats to global human health, including chronic inflammatory diseases, autoimmune disorders, diabetes, vascular diseases and the majority of cancers. Accordingly, the NF-κB pathway is widely considered an attractive therapeutic target in a broad range of malignant and non-malignant diseases. Yet, despite the aggressive efforts by the pharmaceutical industry to develop a specific NF-κB inhibitor, none has been clinically approved, due to the dose-limiting toxicities associated with the global suppression of NF-κB. In this review, we summarise the main strategies historically adopted to therapeutically target the NF-κB pathway with an emphasis on oncology, and some of the emerging strategies and newer agents being developed to pharmacologically inhibit this pathway

Inhibitory feedback control of NF-κB signalling in health and disease.

Cells must adapt to changes in their environment to maintain cell, tissue and organismal integrity in the face of mechanical, chemical or microbiological stress. Nuclear factor-κB (NF-κB) is one of the most important transcription factors that controls inducible gene expression as cells attempt to restore homeostasis. It plays critical roles in the immune system, from acute inflammation to the development of secondary lymphoid organs, and also has roles in cell survival, proliferation and differentiation. Given its role in such critical processes, NF-κB signalling must be subject to strict spatiotemporal control to ensure measured and context-specific cellular responses. Indeed, deregulation of NF-κB signalling can result in debilitating and even lethal inflammation and also underpins some forms of cancer. In this review, we describe the homeostatic feedback mechanisms that limit and 're-set' inducible activation of NF-κB. We first describe the key components of the signalling pathways leading to activation of NF-κB, including the prominent role of protein phosphorylation and protein ubiquitylation, before briefly introducing the key features of feedback control mechanisms. We then describe the array of negative feedback loops targeting different components of the NF-κB signalling cascade including controls at the receptor level, post-receptor signalosome complexes, direct regulation of the critical 'inhibitor of κB kinases' (IKKs) and inhibitory feedforward regulation of NF-κB-dependent transcriptional responses. We also review post-transcriptional feedback controls affecting RNA stability and translation. Finally, we describe the deregulation of these feedback controls in human disease and consider how feedback may be a challenge to the efficacy of inhibitors

Mechanisms of NF-κB p65 and strategies for therapeutic manipulation

The transcription factor NF-κB is a critical regulator of immune and inflammatory responses. In mammals, the NF-κB/Rel family comprises five members: p50, p52, p65 (Rel-A), c-Rel, and Rel-B proteins, which form homo- or heterodimers and remain as an inactive complex with the inhibitory molecules called IκB proteins in resting cells. Two distinct NF-κB signaling pathways have been described: 1) the canonical pathway primarily activated by pathogens and inflammatory mediators, and 2) the noncanonical pathway mostly activated by developmental cues. The most abundant form of NF-κB activated by pathologic stimuli via the canonical pathway is the p65:p50 heterodimer. Disproportionate increase in activated p65 and subsequent transactivation of effector molecules is integral to the pathogenesis of many chronic diseases such as the rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and even neurodegenerative pathologies. Hence, the NF-κB p65 signaling pathway has been a pivotal point for intense drug discovery and development. This review begins with an overview of p65-mediated signaling followed by discussion of strategies that directly target NF-κB p65 in the context of chronic inflammation

Nuclear Import Mechanisms of STAT and NF-kB Transcription Factors

The eukaryotic cell nucleoplasm is separated from the cytoplasm by the nuclear envelope. This compartmentation of eukaryotic cells requires that all nuclear proteins must be transported from the cytoplasm into the nucleus. Transport of macromolecules between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs). Proteins to be targeted into the nucleus by the classical nuclear import system contain nuclear localization signals (NLSs), which are recognized by importin alpha, the NLS receptor. Importin alpha binds to importin beta, which docks the importin-cargo complex on the cytoplasmic side of the NPC and mediates the movement of the complex into the nucleus. Presently six human importin alpha isoforms have been identified. Transcription factors are among the most important regulators of gene expression in eukaryotic organisms. Transcription factors bind to specific DNA sequences on target genes and modulate the activity of the target gene. Many transcription factors, including signal transducers and activators of transcription (STAT) and nuclear factor kB (NF-kB), reside in the cytoplasm in an inactive form, and upon activation they are rapidly transported into the nucleus. In the nucleus STATs and NF-kB regulate the activity of genes whose products are critical in controlling numerous cellular and organismal processes, such as inflammatory and immune responses, cell growth, differentiation and survival. The aim of this study was to investigate the nuclear import mechanisms of STAT and NF-kB transcription factors. This work shows that STAT1 homodimers and STAT1/STAT2 heterodimers bind specifically and directly to importin alpha5 molecule via unconventional dimer-specific NLSs. Importin alpha molecules have two regions, which have been shown to directly interact with the amino acids in the NLS of the cargo molecule. The Arm repeats 2-4 comprise the N-terminal NLS binding site and Arm repeats 7-8 the C-terminal NLS binding site. In this work it is shown that the binding site for STAT1 homodimers and STAT1/STAT2 heterodimers is composed of Arm repeats 8 and 9 of importin alpha5 molecule. This work demonstrates that all NF-kB proteins are transported into the nucleus by importin alpha molecules. In addition, NLS was identified in RelB protein. The interactions between NF-kB proteins and importin alpha molecules were found to be directly mediated by the NLSs of NF-kB proteins. Moreover, we found that p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin alpha3. The results from this thesis work identify previously uncharacterized mechanisms in nuclear import of STAT and NF-kB. These findings provide new insights into the molecular mechanisms regulating the signalling cascades of these important transcription factors from the cytoplasm into the nucleus to the target genes.Tumakalvo erottaa eukaryoottisolun nukleoplasman sytoplasmasta. Tämän vuoksi kaikki sytoplasmassa tuotetut tumaproteiinit kuljetetaan tumakalvolla sijaitsevien tumahuokosten kautta sytoplasmasta tumaan. Tumalokalisaatiosignaalit (nuclear localization signals; NLSs) välittävät tumaan kuljetettavien proteiinien tunnistusta. NLS-signaalin tunnistaa alfa-importiini, NLS-reseptori, joka puolestaan sitoutuu beta-importiiniin. Beta-importiini on kuljetusproteiini, joka välittää tumaan kuljetettavan proteiinin ja alfa-importiinin muodostaman kompleksin sitoutumisen tumahuokoseen ja siirtymisen tumaan. Ihmisellä alfa-importiini-molekyylejä on tunnistettu kuusi erilaista alatyyppiä. Transkriptiotekijät ovat tärkeimpiä geenien ilmentymiseen vaikuttavia molekyylejä. Transkriptiotekijät sitoutuvat tarkoin määrättyihin säätelyjaksoihin DNA-molekyylissä ja vaikuttavat geenien ilmentymiseen. Useat transkriptiotekijät, kuten sytokiinien indusoimat signal transducers and activators of transcription (STAT) ja nuclear factor kB (NF-kB), esiintyvät sytoplasmassa inaktiivisina ja aktivoiduttuaan kulkeutuvat nopeasti tumaan saaden aikaan muutoksia geenien ilmentymisessä. STAT- ja NF-kB-transkriptiotekijät ovat hyvin tärkeitä geenien toiminnan säätelijöitä mm. elimistön puolustusjärjestelmässä viruksia ja mikrobeja vastaan, solujen kehittymisessä ja erilaistumisessa sekä solukuoleman säätelyssä. Tässä työssä selvitimme STAT- ja NF-kB-transkriptiotekijöiden tumakuljetusta. Osoitimme STAT1-homodimeerien ja STAT1/STAT2-heterodimeerien sitoutuvan suoraan alfa5-importiiniin epätavallisten, rakenteellisten NLS-signaalien välityksellä. Alfa-importiinissa on kaksi aluetta, joiden on aiemmin osoitettu suoraan sitoutuvan tumaan kuljetettavien proteiinien NLS-signaalien aminohappoihin. Nämä alueet koostuvat alfa-importiinin N-terminaalisista Arm 2-4 -toistojaksoista, sekä C-terminaalisista Arm 7-8 -toistojaksoista. Selvitimme STAT1-homodimeerien ja STAT1/STAT2-heterodimeerien sitoutuvan alfa5-importiinin Arm 8 ja 9 -toistojaksoihin. Tutkimuksemme osoitti kaikkien NF-kB-proteiinien sitoutuvan suoraan alfa-importiineihin NF-kB-proteiineissa olevien NLS-signaalien välityksellä. Mutaatioanalyysi osoitti p50-proteiinin sitoutuvan alfa3-importiinin N-terminaaliseen Arm 3 -toistojaksoon ja p65-proteiinin C-terminaalisiin Arm 7 - ja Arm 8 -toistojaksoihin. Lisäksi tunnistimme RelB-proteiinista NLS-signaalin. Tässä väitöskirjatyössä identifioimme aiemmin tuntemattomia STAT- ja NF-kB-transkriptiotekijöiden tumakuljetustapahtumia. Tämä väitöskirjatyö syventää tietämystä molekylaarisista mekanismeista signaalinvälitysreiteissä, jotka välittävät STAT- ja NF-kB-transkriptiotekijöiden siirtymistä sytoplasmasta tumaan kohdegeenien säätelyalueille

IκBα inhibits apoptosis at the outer mitochondrial membrane through a novel, NF-κB–independent, interaction with VDAC1

The inducible transcription factor NF-κB is tightly regulated by the inhibitory IκB-family of proteins that associate with the transcription factor and act in response to stress stimuli. The best studied inhibitory protein is IκBα which resides in the cytosol where it retains NF-κB. Our study shows that IκBα also associates with the outer mitochondrial membrane (OMM) and exerts an unexpected novel anti-apoptotic function, independent of NF-κB inhibition. IκBα-/- cells become refractory to apoptosis when IκBα is specifically reconstituted at the OMM. We found that cancer cells with constitutively active NF-κB accumulate IκBα at the OMM and when its expression is down-regulated these cells are sensitised to apoptosis. At the OMM IκBα associates with VDAC1 and hexokinase II (HKII). Our findings show that IκBα inhibits the dissociation of HKII from VDAC1 and prevents Bax-mediated cytochrome c release. Deletion mutants of IκBα reveal a domain necessary for apoptosis inhibition that is different from the domain for NF-κB retention, thereby separating the two functions. These results reveal an unexpected activity of IκΒα in guarding the integrity of the OMM against apoptosis induction and open possibilities for more specific interference in diseases involving deregulated NF-κB