Reconstruction and analysis of the NF-kB pathway interactome: a systems biology approach

Background. One of the phenomena observed in human aging is the progressive increase of a systemic inflammatory state, a condition referred to as “inflammaging”, negatively correlated with longevity. A prominent mediator of inflammation is the transcription factor NF-kB, that acts as key transcriptional regulator of many genes coding for pro-inflammatory cytokines. Many different signaling pathways activated by very diverse stimuli converge on NF-kB, resulting in a regulatory network characterized by high complexity. NF-kB signaling has been proposed to be responsible of inflammaging. Scope of this analysis is to provide a wider, systemic picture of such intricate signaling and interaction network: the NF-kB pathway interactome. Methods. The study has been carried out following a workflow for gathering information from literature as well as from several pathway and protein interactions databases, and for integrating and analyzing existing data and the relative reconstructed representations by using the available computational tools. Strong manual intervention has been necessarily used to integrate data from multiple sources into mathematically analyzable networks. The reconstruction of the NF-kB interactome pursued with this approach provides a starting point for a general view of the architecture and for a deeper analysis and understanding of this complex regulatory system. Results. A “core” and a “wider” NF-kB pathway interactome, consisting of 140 and 3146 proteins respectively, were reconstructed and analyzed through a mathematical, graph-theoretical approach. Among other interesting features, the topological characterization of the interactomes shows that a relevant number of interacting proteins are in turn products of genes that are controlled and regulated in their expression exactly by NF-kB transcription factors. These “feedback loops”, not always well-known, deserve deeper investigation since they may have a role in tuning the response and the output consequent to NF-kB pathway initiation, in regulating the intensity of the response, or its homeostasis and balance in order to make the functioning of such critical system more robust and reliable. This integrated view allows to shed light on the functional structure and on some of the crucial nodes of thet NF-kB transcription factors interactome. Conclusion. Framing structure and dynamics of the NF-kB interactome into a wider, systemic picture would be a significant step toward a better understanding of how NF-kB globally regulates diverse gene programs and phenotypes. This study represents a step towards a more complete and integrated view of the NF-kB signaling system.

Identificazione di fattori trascrizionali associati al differenziamento neurale in cellule di sarcoma di Ewing: ruolo di NF-kB

Il sarcoma di Ewing (ES) è un tumore maligno pediatrico dell’apparato scheletrico; è associato a una traslocazione specifica codificante la proteina di fusione EWS-FLI1 e all’alta espressione di CD99, una glicoproteina di membrana fisiologicamente coinvolta in diversi processi biologici. EWS-FLI1 e CD99, sono riportati avere ruoli divergenti nella modulazione della malignità e del differenziamento di ES. CD99 inoltre è riportato modulare il pathway di MAPK, il quale interagendo con molteplici fattori di trascrizione partecipa a processi di proliferazione e differenziamento. In questo studio abbiamo investigato in due linee cellulari di ES silenziate per CD99 (TC-71shCD99 e IOR/CARshCD99) l’attività basale di diversi fattori trascrizionali quali: NF-kBp65, AP1, Elk-1, E2F e CREB. L’unico fattore trascrizionale statisticamente significativo è risultato essere NF-kBp65 e abbiamo valutato il suo ruolo nel differenziamento neurale di cellule di ES e la relazione con EWS-FLI1 e CD99. L’attività trascrizionale di NF-kB è stata valutata attraverso gene reporter assay in linee cellulari di ES a diversa espressione di CD99, EWS-FLI1 e NF-kB stesso. Il differenziamento neurale è stato valutato come espressione di βIII-Tubulin in immunofluorescenza. Il silenziamento di CD99 induce una down-modulazione dell’attività trascrizionale di NF-kB, mentre il knockdown di EWS-FLI1 ne induce un’aumento. Inoltre, il silenziamento di EWS-FLI1 non è in grado di contrastare la riduzione dell’attività di NF-kB osservata dopo silenziamento di CD99, suggerendo un ruolo dominante del CD99 nel signaling di NF-kB. Cellule deprivate di CD99 ma non di EWS-FLI1, mostrano un fenotipo differenziato in senso neurale, fenotipo che viene perso quando le cellule sono indotte a sovraesprimere NF-kB. Inoltre, in cellule CD99 positive, il silenziamento di NF-kB induce un leggero differenziamento neurale. In conclusione, questi dati hanno evidenziato il ruolo di NF-kB nel differenziamento di cellule di ES e che potrebbe essere un potenziale target nel ridurre la progressione di questo tumore.

Studio del pathway della Displasia Ectodermica Anidrotica (EDA)

Anhidrotic Ectodermal Dysplasia (EDA), is the most frequent form among Ectodermal Dysplasias, hereditary genetic disorders causing ectodermal appendages defective development. Indeed, EDA is characterized by defective formation of hair follicles, sweat glands and teeth both in human patients and animals. EDA, the gene mutated in Anhidrotic Ectodermal Dysplasia, encodes Ectodysplasin, a TNF family member that activates NF-kB mediated transcription. This disease can occur with mutations in other EDA-NF-kB pathway members, as EDA receptor, EDAR and its adapter, EDARADD. Moreover, mutations in TRAF6, NEMO, IKB and NF-kBs genes are responsible for Immunodeficiency associated EDA (EDA-ID). Several molecules, as SHH, WNT/DKK, BMP and LTβ, have already been reported to be EDA pathway regulators or effectors although the knowledge of the full spectrum of EDA targets remains incomplete. During the first part of the research project a gene expression analysis was performed in primary keratinocytes from Wild-type and Tabby (EDA model mouse) mice to identify novel EDA target genes. Earlier expression profiling at various developmental time points in Tabby and Wild-type mouse skin reported genes differentially expressed in the two samples and, to increase the resolution to find genes whose expression may be restricted to epidermal cells, the study was extended to primary keratinocyte cultures established from E19 Wild-type and Tabby skin. Using microarrays bearing 44,000 gene probes, we found 385 “preliminary candidate” genes whose expression was significantly affected by Eda defect. By comparing expression profiles to those from Eda-A1 (where Eda-A1 is highly expressed) transgenic skin, we restricted the list to 38 “candidate EDA targets”, 14 of which were already known to be expressed in hair follicles or epidermis. This work confirmed expression changes for 3 selected genes, Tbx1, Bmp7, and Jag1, both in primary keratinocytes and in Wild-type and Tabby whole skin, by Q-PCR and Western blotting analyses. Thus, this study detected novel candidate pathways downstream of EDA. In the second part of the research project, plasmid constructs were produced and analyzed to create a transgenic mouse model for Immunodeficiency associated EDA disease (XL-EDA-ID). In particular, plasmids containing mouse Wild-type and mutated Nemo cDNA under K-17 epidermis-specific promoter control and a Flag tag, were prepared, on the way to confine transgene expression to mice epidermis and to determine EDA phenotype without immunodeficiency for a comparison to Tabby model phenotype. EDA-ID mutations reported in patients and selected for this study are: C417R (C409R in mouse), causing Zinc Finger protein domain destabilization and A288G (A282G in mouse) affecting oligomerization of the protein. Moreover, the ex-novo mutation, ZnF, C-terminal Zinc Finger domain deletion, was tested. Thus, the constructs were analyzed by transient transfection, Western blotting and luciferase assays techniques, detecting Nemo Wild-type and mutant protein products and residue NF-kB activity in presence of mutants, after TNF stimulation. In particular, MEF_Nemo-/- cell line was used to monitor NF-kB activity without endogenous Nemo gene. Results show reduced NF-kB activity in presence of mutated Nemo forms compared to Wild-type: 81% for A282G (A288G in human); 24% for C409R (C417R in human); 15% for ZnF. C409R mutation (C417R in human), reported in 6 EDA-ID human patients, was selected to prepare transgenic model mouse. Mice (white, FVP) born following K17-promoter-Flag-Nemo_C409R plasmid region pronuclear injection, were analyzed for the transgene presence in the genotype and a preliminar examination of their phenotype was performed. In particular, one mouse showed considerable coat defects if compared to Wild-type mice. This preliminar analysis suggests a possible influence of Nemo mutant over-expression in epidermis without immunodeficiency. Still, more microscopic studies to analyze hair subtypes, Guard, Awl and Zigzag (usually alterated inTabby mouse model), Immunohistochemistry experiments to detect epidermis restricted Nemo expression and sweat glands analysis, will follow. This and other transgene positive mice will be crossed with black mice C57BL6 to obtain at least two indipendent agouti lines to analyze. Theses mice will be used in EDA target genes detection through microarrays. Following, plasmid constructs containing other Nemo mutant forms (A282G and ZnF) might be studied by the same experimental approaches to prepare more transgenic model mice to compare to Nemo_C409R and Tabby mouse models.

Analisi funzionale dei recettori per le neurotrofine p75NTR e Trka in neuroblastoma

The biological complexity of NGF action is achieved by binding two distinct Neurotrophin receptors, TrkA and p75NTR. While several reports have provided lines of evidence on the interaction between TrkA and p75NTR at the plasma membrane, much fewer data are available on the consequence of such an interaction in terms of intracellular signaling. In this study, we have focused on how p75NTR may affect TrkA downstream signaling with respect to neuronal differentiation. Here, we have shown that cooperation between p75NTR and TrkA results in an increased NGF-mediated TrkA autophosphorylation, leads to a sustained activation of ERK1/2 and accelerates neurite outgrowth. Interestingly, neurite outgrowth is concomitant with a selective enhancement of the AP-1 activity and the transcriptional activation of genes such as GAP-43 and p21(CIP/WAF), known to be involved in the differentiation process. Collectively, our results unveil a functional link between the specific expression profile of neurotrophin receptors in neuronal cells and the NGF-mediated regulation of the differentiation process possibly through a persistent ERKs activation and the selective control of the AP-1 activity. In our studies we discuss the functional role of the neurotrophin receptor p75NTR and TrkA in a ligand-dependent signal transduction. It is known that p75NTR is also involved in the mediation of cell death ligand dependent. Here we show for the first time that the membrane receptor p75NTR, upon binding to b- Amyloid (Ab) peptide, is able to transduce a cytotoxic signal through a mechanism very similar to the one adopted by Tumor Necrosis Factor Receptor 1 (TNFR1), when activated by TNFa. We define that in neuroblastoma cell line Ab cytotoxicity signals through a pathway depending on p75NTR death domain (DD), mostly through some specific conserved residues. We identified that TRADD is the first interactor recruiting to the membrane and activates JNK and NF-kB transcription factors. Since Ab is defined as the most important aetiologic element associated with the Alzheimer’s Disease (AD), characterization of the mechanism involved in the mediation of the neurodegeneration can suggest also new therapeutic approaches.

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

Analisi genetico-molecolare dei linfomi a cellule T periferiche

Molecular profiling of Peripheral T-cell lymphomas not otherwise specified Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of tumors that the WHO classification basically subdivides into specified and not otherwise specified (NOS). In Western countries, they represent around 12% of all non-Hodgkin's lymphomas. In particular, PTCL/NOS is the commonest subtype, corresponding to about 60-70% of all T-cell lymphomas. However, it remains a complex entity showing great variety regarding either morphology, immunophenotype or clinical behavior. Specially, the molecular pathology of these tumors is still poorly known. In fact, many alteration were found, but no single genes were demonstrated to have a pathogenetic role. Recently, gene expression profiling (GEP) allowed the identification of PTCL/NOS-associated molecular signatures, leading to better understanding of their histogenesis, pathogenesis and prognostication. Interestingly, proliferation pathways are commonly altered in PTCLs, being highly proliferative cases characterized by poorer prognosis. In this study, we aimed to investigate the possible role in PTCL/NOS pathogenesis of selected molecules, known to be relevant for proliferation control. In particular, we analyzed the cell cycle regulators PTEN and CDKN1B/p27, the NF-kB pathway, and the tyrosin kinase PDGFR. First, we found that PTEN and p27 seem to be regulated in PTCL/NOS as in normal T-lymphocytes, as to what expression and cellular localization are concerned, and do not present structural abnormalities in the vast majority of PTCL/NOS. Secondly, NF-kB pathway appeared to be variably activated in PTCL/NOS. In particular, according to NF-kB gene expression levels, the tumors could be divided into two clusters (C1 and C2). Specially, C1 corresponded to cases presenting with a global down-regulation of the entire pathway, while C2 showed over-expression of genes involved in TNF signaling. Notably, by immunohistochemistry, we showed that either the canonical or the alternative NK-kB pathway were activated in around 40% of cases. Finally, we found PGDFRA to be consistently over-expressed (at mRNA and protein level) and activated in almost all PTCLs/NOS. Noteworthy, when investigating possible causes for PDGFRA deregulation, we had evidences that PDGFR over-expression is due to the absence of miR-152, which appeared to be responsible for PDGFRA silencing in normal T-cells. Furthermore, we could demonstrate that its aberrant activation is sustained by an autocrine loop. Importantly, this is the first case, to the best of our knowledge, of hematological tumor in which tyrosin kinase aberrant activity is determined by deregulated miRNA expression and autocrine loop activation. Taken together, our results provide novel insight in PTCL/NOS pathogenesis by opening new intriguing scenarios for innovative therapeutic interventions.

Alimenti Funzionali e Componenti Nutraceutici come Biomodulatori

Recent knowledge supports the hypothesis that, beyond meeting nutrition needs, diet may modulate various functions in the body and play beneficial roles in some diseases. Research on functional foods is addressing the physiologic effects and health benefits of foods and food components, with the aim of authorizing specific health claims. The recognition that oxidative stress plays a major role in the pathophysiology of cardiac disorders has led to extensive investigations of the protective effects of exogenous antioxidants, but results are controversial. A promising strategy for protecting cardiac cells against oxidative damage may be through the induction of endogenous phase 2 enzymes with the enhancement of cellular antioxidant capacity. Sulforaphane (SF), a naturally occurring isothiocyanate abundant in Cruciferous vegetables, has gained attention as a potential chemopreventive compound thanks to its ability to induce several classes of genes implicated in reactive oxygen species (ROS) and electrophiles detoxification. Antioxidant responsive element (ARE)-mediated gene induction is a pivotal mechanism of cellular defence against the toxicity of electrophiles and ROS. The transcription factor NF-E2-related factor-2 (Nrf2), is essential for the up-regulation of these genes. We investigated whether SF could exert cardioprotective effects against oxidative stress and elucidated the mechanisms underpinning these effects. Accordingly, using cultured rat neonatal cardiomyocytes as a model system, we evaluated the time-dependent induction of gene transcription, the corresponding protein expression and activity of various antioxidant and phase 2 enzymes (catalase, superoxide dismutase, glutathione and related enzymes glutathione reductase, glutathione peroxidase and glutathione S-transferase, NAD(P)H: quinone oxidoreductase 1 and thioredoxine reductase) elicited by SF. The results were correlated to intracellular ROS production and cell viability after oxidative stress generated by H2O2, and confirmed the ability of SF to exert cytoprotective effects acting as an indirect antioxidant. Furthermore, to get better insight into SF mechanism of action, we investigated the effect of SF treatment on Nrf2 and the upstream signalling pathways MAPK ERK1/2 and PI3K/Akt, known to mediate a pro survival signal in the heart. The use of specific inhibitors of ERK1/2 and Akt phosphorylation demonstrated their involvement in phase 2 enzymes induction. The concentration of SF tested in this study is comparable to peak plasma concentration achieved after dietary exposure giving clear relevance to our data to support dietary intake of Cruciferous vegetables in cytoprotection against oxidative stress, a common determinant of many cardiovascular diseases.