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.

Sistemi endorfinergici e modulazione di segnali molecolari e profili trascrizionali coinvolti in processi di protezione e autoriparazione del miocardio danneggiato

Con il termine IPC (precondizionamento ischemico) si indica un fenomeno per il quale, esponendo il cuore a brevi cicli di ischemie subletali prima di un danno ischemico prolungato, si conferisce una profonda resistenza all’infarto, una delle principali cause di invalidità e mortalità a livello mondiale. Studi recenti hanno suggerito che l’IPC sia in grado di migliorare la sopravvivenza, la mobilizzazione e l’integrazione di cellule staminali in aree ischemiche e che possa fornire una nuova strategia per potenziare l’efficacia della terapia cellulare cardiaca, un’area della ricerca in continuo sviluppo. L’IPC è difficilmente trasferibile nella pratica clinica ma, da anni, è ben documentato che gli oppioidi e i loro recettori hanno un ruolo cardioprotettivo e che attivano le vie di segnale coinvolte nell’IPC: sono quindi candidati ideali per una possibile terapia farmacologica alternativa all’IPC. Il trattamento di cardiomiociti con gli agonisti dei recettori oppioidi Dinorfina B, DADLE e Met-Encefalina potrebbe proteggere, quindi, le cellule dall’apoptosi causata da un ambiente ischemico ma potrebbe anche indurle a produrre fattori che richiamino elementi staminali. Per testare quest’ipotesi è stato messo a punto un modello di “microambiente ischemico” in vitro sui cardiomioblasti di ratto H9c2 ed è stato dimostrato che precondizionando le cellule in modo “continuativo” (ventiquattro ore di precondizionamento con oppioidi e successivamente ventiquattro ore di induzione del danno, continuando a somministrare i peptidi oppioidi) con Dinorfina B e DADLE si verifica una protezione diretta dall’apoptosi. Successivamente, saggi di migrazione e adesione hanno mostrato che DADLE agisce sulle H9c2 “ischemiche” spronandole a creare un microambiente capace di attirare cellule staminali mesenchimali umane (FMhMSC) e di potenziare le capacità adesive delle FMhMSC. I dati ottenuti suggeriscono, inoltre, che la capacità del microambiente ischemico trattato con DADLE di attirare le cellule staminali possa essere imputabile alla maggiore espressione di chemochine da parte delle H9c2.

Discovery and Pharmacological Characterization of a Novel Small Molecule Antagonist of Integrin α4β1: Focus on Antiallergic Activity

Allergy is a common hypersensitivity disorder that affects 15% to 20% of the population and its prevalence is increasing worldwide. Its severity correlates with the degree of eosinophil infiltration into the conjunctiva, which is mediated by chemokines that stimulate the production of adhesion molecules like intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the endothelial cell surface. The α4β1 and α4β7 integrins are expressed in eosinophils and contribute to their activation and infiltration in AC through the binding to VCAM-1 or fibronectin, expressed on vascular endothelial cells. Blockade of α4 integrins might be a therapeutical achievement in allergic eye diseases. DS 70, that show an IC50 in the nanomolar range against α4β1 integrin in Jurkat cells and in the eosinophilic cell line EOL-1. This compound was able to prevent cell adhesion to VCAM-1 and FN in vitro. In a scintillation proximity assay DS70 displaced 125I-FN binding to human α4β1 integrin and, in flow cytometry analysis, it antagonized the binding of a primary antibody to α4β1 integrin expressed on the Jurkat cells surface as well. Furthermore, we analysed also its effects on integrin α4β1 signalling. In an vivo model of allergic conjunctivitis, topical DS70 reduced the clinical aspects of EPR (early phase reaction) and LPR (late phase reaction), by reducing clinical score, eosinophil accumulation, mRNA levels of cytochines and chemochines pro-inflammatory and the conjunctival expression of α4 integrin. In conclusion, DS70 seems a novel antiallergic ocular agent that has significant effects on both early and late phases of ocular allergy.

Studio delle citochine nella distrofia muscolare di Emery-Dreifuss: Possibili marker patogenetici e bersagli di cura della malattia

La distrofia muscolare di Emery-Dreifuss (EDMD) è una miopatia degenerativa ereditaria caratterizzata da debolezza e atrofia dei muscoli senza coinvolgimento del sistema nervoso. Individui EDMD presentano, inoltre, cardiomiopatia con difetto di conduzione che provoca rischio di morte improvvisa. Diversi studi evidenziano un coinvolgimento di citochine in diverse distrofie muscolari causanti infiammazione cronica, riassorbimento osseo, necrosi cellulare. Abbiamo effettuato una valutazione simultanea della concentrazione di citochine, chemochine, fattori di crescita, presenti nel siero di un gruppo di 25 pazienti EDMD. L’analisi effettuata ha evidenziato un aumento di citochine quali IL-17, TGFβ2, INF-γ e del TGFβ1. Inoltre, una riduzione del fattore di crescita VEGF e della chemochina RANTES è stata rilevata nel siero dei pazienti EDMD rispetto ai pazienti controllo. Ulteriori analisi effettuate tramite saggio ELISA hanno evidenziato un aumento dei livelli di TGFβ2 e IL-6 nel terreno di coltura di fibroblasti EDMD2. Per testare l’effetto nei muscoli, di citochine alterate, abbiamo utilizzato terreno condizionante di fibroblasti EDMD per differenziare mioblasti murini C2C12. Una riduzione del grado di differenziamento è stata osservata nei mioblasti condizionati con terreno EDMD. Trattando queste cellule con anticorpi neutralizzanti contro TGFβ2 e IL-6 si è avuto un miglioramento del grado di differenziamento. In C2C12 che esprimevano la mutazione H222P del gene Lmna,non sono state osservate alterazioni di citochine e benefici di anticorpi neutralizzanti. I dati mostrano un effetto patogenetico delle citochine alterate come osservato in fibroblasti e siero di pazienti, suggerendo un effetto sul tessuto fibrotico di muscoli EDMD. Un effetto intrinseco alla mutazione della lamina A è stato rilevato sul espressione di caveolina 3 in mioblasti differenziati EDMD. I risultati si aggiungono a dati forniti sulla patogenesi dell' EDMD confermando che fattori intrinseci ed estrinseci contribuiscono alla malattia. Utilizzo di anticorpi neutralizzanti specifici contro fattori estrinseci potrebbe rappresentare un approccio terapeutico come mostrato in questo studio.

Interactions between the gut microbiota, short-chain fatty acids and the immune system in pediatric patients undergoing hematopoietic stem cell transplantation

The gut microbiota (GM) is essential for human health and contributes to several diseases; indeed it can be considered an extension of the self and, together with the genetic makeup, determines the physiology of an organism. In this thesis has been studied the peripheral immune system reconstitution in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (aHSCT) in the early phase; in parallel, have been also explored the gut microbiota variations as one of the of primary factors in governing the fate of the immunological recovery, predisposing or protecting from complications such as the onset of acute graft-versus-host disease (GvHD). Has been demonstrated, to our knowledge for the first time, that aHSCT in pediatric patients is associated to a profound modification of the GM ecosystem with a disruption of its mutualistic asset. aGvHD and non-aGvHD subjects showed differences in the process of GM recovery, in members abundance of the phylum Bacteroidetes, and in propionate fecal concentration; the latter are higher in the pre-HSCT composition of non-GvHD subjects than GvHD ones. Short-chain fatty acids (SCFAs), such as acetate, butyrate and propionate, are end-products of microbial fermentation of macronutrients and distribute systemically from the gut to blood. For this reason, has been studied their effect in vitro on human DCs, the key regulators of our immune system and the main player of aGvHD onset. Has been observed that propionate and, particularly, butyrate show a strong and direct immunomodulatory activity on DCs reducing inflammatory markers such as chemokines and interleukins. This study, with the needed caution, suggests that the pre-existing GM structure can be protective against aGvHD onset, exerting its protective role through SCFAs. They, indeed, may regulate cell traffic within secondary lymphoid tissues, influence T cell development during antigen recognition, and, thus, directly shape the immune system.