Literatura académica sobre el tema "Recettore di membrana"

Lo scompenso cardiaco (SC), processo evolutivo comune di più malattie cardiovascolari a differente eziologia (ad es. infarto del miocardio, ipertensione, cardiomiopatie, disturbi valvolari e altre), è diventato sempre più comune nella popolazione anziana, influenzando drasticamente il tasso di sopravvivenza e la qualità della vita. L’iperattività del sistema nervoso simpatico (SNS) che si associa allo SC determina un aumento delle catecolamine circolanti epinefrina e norepinefrina che, attraverso l’attivazione dei recettori beta-adrenergici (β-AR), svolgono un ruolo critico nella regolazione della funzione del sistema cardiovascolare. Una caratteristica distintiva dello SC è la diminuzione o la desensibilizzazione dei recettori β1-adrenergici (β1-AR) sulla membrana delle cellule cardiache. Le catecolamine e lo stress ossidativo sono coinvolti nella regolazione della densità dei β-AR. Lo stress ossidativo associato alla disfunzione mitocondriale sembra giocare un ruolo importante nella fisiopatologia dello SC. Infatti, una condizione di stress ossidativo è stata osservata sia in pazienti con SC che in modelli animali, e un’eccessiva esposizione a specie reattive dell’ossigeno (ROS) diminuisce l’espressione di β1-AR in cardiomiociti murini, sebbene i meccanismi sottostanti rimangano ancora non chiari. Recentemente, è stato scoperto che il recettore periferico delle benzodiazepine (PBR) svolge un ruolo chiave oltre che nell’energetica cellulare, nella regolazione della fisiologia mitocondriale e dell’equilibrio redox nei cardiomiociti. Nel presente studio, abbiamo valutato gli effetti delle catecolamine e dei ligandi del PBR sulla densità dei β1- e β2-AR nei monociti umani isolati da sangue periferico, che sono noti per esprimere entrambi i β-AR. La densità dei β-AR è stata misurata mediante citometria a flusso utilizzando anticorpi selettivi diretti contro un epitopo extracellulare di β1-AR o β2-AR. Il trattamento dei monociti con benzodiazepine induceva una riduzione della densità del β1-AR, ma non del β2-AR, sulla membrana dei monociti che veniva ripristinata utilizzando [1-(2-chlorophenyl)-N-methyl-(1-meth-ylpropyl)-3 isoquinolinecarboxamide] (PK11195), un antagonista del PBR. Questi risultati suggeriscono un possibile ruolo del PBR nella regolazione della densità del β1-AR proponendo i monociti isolati dal sangue periferico sia come modello in vitro utile per lo studio del sistema recettoriale β-adrenergico che come potenziali biomarcatori di progressione della malattia e risposta alla terapia.

After some words on the scientific role of Professor Paolo Mantegazza atthe University of Milan (4, 5, 6), I briefly illustrate some studies related to the occurrence of neurotransmitter and receptor re-specification in the adult animals. The greatdiscoveries of the early twentieth century on neuronal communication have established that the majority of communication between nerve cells occurs through a special structure, the synapse, allowing the one-way transfer of information between twocells through the release of a neurotransmitter from the presynaptic cell and its recognition by receptors localized in the postsynaptic cell. According to H. Dale axiom (9) each neuron could be identified on the basis of the neurotransmitter released and theinnervated cell by the type of receptors expressed; then neurons could be classified asexcitatory if they release acetylcholine, glutamate or other transmitters, or inhibitory ifthey release GABA or glycine. However, in recent years many studies have shown that, especially during development, a neuron could release and co-release several neuro-transmitters, sometimes even simultaneously, changing its classification from excitatory to inhibitory and vice versa(7). This researches opened a new field of study onsynaptic plasticity: the neurotransmitter and receptor re-specification. Our group, together with Prof. Mantegazza, tried to “force” it through experiments of denervation and heterologous re-innervation in the autonomic nervous system and at the neuromuscular junction. In a first series of experiments we studied the regenerative capabilities of the peripheral nervous system in three experimental models: a) re-innervation of the denervated superior cervical ganglion (SCG) (14, 15, 22) by cholinergicefferent vagal fibers, b) re-innervation of peripheral effectors smooth muscles (nicti-tating membrane) by the cholinergic preganglionic fibers; c) re-innervation in an in vivo transplant model of peripheral organs by the SCG. In these researches we haveestablished: 1) that a sympathetic ganglion could be re-innervated by vagal fibersforming normal ganglionic synapses, but with a strong reshaping, in vivo, of the cen-tral neural circuits so that sympathetic stimuli occurred through a vagal excitation; 2) preganglionic cholinergic fibers innervate the smooth muscle of the nictitating mem-brane releasing catecholamines instead of acetylcholine; 3) that in an in vivo model ofSCG transplant together with iris or adrenal medulla fragments, the SCG was able todistinguish between organs that required a postsynaptic innervation, iris, which wasinnervated, and organs that require a presynaptic innervation, the adrenal medulla,that was not innervated. We were then in the presence, even in the adult animal, of anew nervous plasticity with re-specification the neurotransmitter. These resultsdemonstrate that heterologous innervation could “force” plasticity in adult peripheralnervous system, alters the biological properties of neurons, upsets central neuronal circuits, but continues to maintain in experimental transplants basic rules of innervation between neurons and peripheral organs. Thirty years later, the group of prof. Brunelliin Brescia (23), along with pharmacologists and physiologists, had highlighted the pos-sibility of re-innervate striated muscles in a functional way with nerve fibers derivedfrom the red nucleus of the vestibular complex. The interest was, once again, in thefact that the re-innervating fibers were of glutamatergic type, and not cholinergic likethose of normal motor neurons, and that neuromuscular transmission was transformedfrom nicotinic cholinergic in glutamatergic. A new type of plasticity: the receptor re-specification had occurred also in this experimenal model. In close cooperationbetween our Milan and the Brescia group we could reconfirm with more appropriateexperiments that the re-innervation occurred; that neuromuscular junction had a glutamatergic transmission; that new re-innervating fibers made synapses at the same sitesof the previous neuromuscular junctions; that the new fibers release glutamate; andthat muscle cells expressed new glutamate receptors (24). Once again we were in thepresence of an extraordinary phenomenon of synaptic plasticity, in this case a receptorre-specification, and again with a strong impact on the central nervous system circuits.These experiences, along with many others now available in the literature, show thatthe adult peripheral nervous system, both autonomous and musculoskeletal, has aplasticity unthinkable before and open a field of great interest aiming at the understanding how neuronal specificity is regulated and at the investigation of non-canonical, but perhaps functional, re-innervation experiments in transplants and in post-traumatic surgery.

T lymphocytes are activated when their T-cell receptors (TCRs) recognize and interact with the specific antigenic complexes formed by antigen-derived peptides bound to proteins of the major histocompatibility complex (MHC) and exposed on the surface of an antigen-presenting cell (APC). The contact site between these two cells is referred as immunological synapse (IS) and represents a highly specialized cellular junction, where T lymphocyte receives and integrates several signals provided by cellular partner, in order to choose between tolerance and immunity. These signals, additional to TCR, are substantially provided by costimulatory molecules that are tuned by inflammatory environment, thus coding the context of antigenic presentation. The immune system has developed different strategies for such a complex function and recently new costimulatory mechanisms, different from the classical ones based on membrane receptors, have been identified, such as signal amplification by membrane rafts and costimulation through chemokines. In this thesis I have been interested in defining the molecular mechanisms underlying these new costimulatory strategies at the IS. The cellular plasma membrane contains small, heterogeneous and highly dynamic, microdomains, enriched in sterol and sphingolipid and in selective proteins, defined as membrane rafts. In T-cell plasma membrane these microdomains are recruited into the IS, forming a platform where TCR signal is protected and amplified, and thus contribute to costimulation. In our group it has been proposed that CD28, the main costimulatory molecule for naive T lymphocytes, amplifies TCR signal by inducing membrane rafts rearrangement and recruitment into the IS. In order to identify the molecular mechanism allowing CD28-mediated rafts recruitment into the IS, and considering the essential role played by actin cytoskeleton in molecule mobilization toward IS, we focused on interaction among CD28, cytoskeleton and rafts. In this thesis it is demonstrated that CD28 binds to filamin-A (FLNa), an actin-binding protein able to induce actin crosslinking and to stabilize the cortical cytoskeleton, and recruits FLNa into the IS. The interaction between CD28 and FLNa, as well as the recruitment of FLNa into the IS, require the same CD28 prolin-rich motif needed for membrane rafts mobilization into the IS. Moreover FLNa silencing by small interference (si)RNA inhibits CD28-induced rafts recruitment into the IS, Cdc42 activation (that regulates cytoskeletal rearrangements) and CD28 costimulation. These results indicate that CD28 uses FLNa to integrate signalling pathways, resulting in actin crosslinking and lipid raft recruitment into the IS, thus sustaining TCR signaling and lowering the T-cell activation threshold. The costimulatory properties of chemokines have been recently demonstrated in human T lymphocytes. In these cells, the chemokine receptor CXCR4 is constitutively expressed and regulates lymphocyte migration towards gradients of CXCL12; in contrast, CCR5 is expressed only in activated T cells and leads their migration towards gradients of CCL3, CCL4 and CCL5. These two receptors are involved in several pathological conditions, such as autoimmunity, cancer and HIV (human immunodeficiency virus). Our group has demonstrated that during T-cell stimulation both CXCR4 and CCR5 are recruited and trapped into the IS, through a mechanism that requires chemokine secretion by APC. The CXCR4 and CCR5 recruitment into the IS results in stronger interactions between T cell and APC, in reduced responsiveness to chemotactic gradients and in higher levels of T-cell proliferation and IFN-? (interferon-?) production. Interestingly, we found that during T cell activation chemokine receptors are coupled with Gq instead of Gi, the classical G protein coupled to these receptors during cell migration. The aim of my thesis was to study the mechanism for CXCR4 and CCR5 versatility in function and signaling, and to identify the requirements for chemokine-induced T-cell costimulation. Since chemokine receptors can form receptor complexes with specific pharmacological and signaling properties through homo- and hetero-dimerization, we hypothesized that molecular complexes between CXCR4 and CCR5 in T lymphocytes are required for their costimulation at the IS. In this thesis it is demonstrated that, in contrast with CXCR4 and CCR5 chemotactic functions, which depends on receptor homodimers, the costimulatory function of these receptors requires their functional collaboration: CXCR4 and CCR5 must be co-recruited into the IS and must be co-expressed by T cell to costimulate cytokine production. Moreover it has been demonstrated that co-expressed CXCR4 and CCR5 form constitutive complexes (hetero-dimers or hetero-oligomers), suggesting that cooperation between receptors represents one key strategy for the functional plasticity of chemokines. In conclusion, my study on novel T-cell costimulation mechanisms highlight the complexity of the process leading to transmission of signals at the IS. This integrated and dynamic process involves soluble mediators, membrane receptors and the cell cytoskeleton, and generates micro-environments specific for signal amplification by locally modifying the cell membrane composition and its signaling complexes.

The tyrosine phosphorylation in eukaryotic proteins is a key event for transducing enviromental cues into cellular responses ranging from cell-to-cell communication to proliferation, differentiation, cell death and survival. This mechanism of signal transduction is mediated by the opposing and concerted action of Protein Tyrosin Kinases (PTKs), which add phosphoryl groups to target proteins, and Protein Tyrosin Phosphatases (PTPs), which remove them; both classes of enzymes can take part in activatory and inhibitory signalling processes. Tyrosin phosphorylation calls also into action the Src-homology 2 domain (SH2), which is contained in a myriad of proteins with varied functions and that directs protein-protein interaction by “sensing” the phosphorylated state of tyrosine residues, taking part in the modulation of signal transduction. This three-part system has recently been baptized as the “writer “ (PTK), “eraser” (PTP) and “reader” (SH2) tollkit, emphasizing how the combination and dynamic interplay of the elements can generate diverse and complex regulatory outputs. The aim of this work was to determine how the tyrosine phosphorylation is involved in the cellular response to extracellular signals; we investigated the events which trigger an altered pathway in diseases, mainly the role of non-receptor PTPs in three different pathological conditions. In Polycytemia Vera (PV) and Essential Thrombocythemia (ET), which are Philadelphia-negative myeloproliferative disorders (Ph-MPDs), our results demonstrate that in resting platelets the dephosphorylation of the Src-Tyr527 is due the SHP-2 constitutive activity, a non-receptor PTPs, leading to the Src preactivation. The anomalous activation of the kinase is implicated in the hypersensivity of Ph-MPDs and likely involved in the functional abnormalities of PV and ET platelets. In Heparin-Induced-Thrombocytopenia (HIT), immunological reaction that lead to the activation of FcγRIIA in platelets, we have not identified any correlation between the polymorphisms of receptor and HIT, but our results demonstrate that the phosphorylation of FcγRIIA-ITAM motif is due the type of ligand and that influences the responses in the platelets. We stimulated normal platelets with either IV.3, functional blocking antibody against FcγRIIA, or complexes of IgG molecules, and we have not highlighted phosphorylation of the ITAM or aggregation, but we observed a mildly shape change. Interestingly, the pre-incubation of the platelets with PTP-1B, the largest PTPs in platelets, in association with IV.3 and IgG as agonists, leads to phosphorylation of the ITAM and to aggregation. This mechanism may be involved in the clearance of IgG-containing complexes from the circulation by platelets. Finally, we demonstrate for the first time that the PDGF (platelet-derived growth factor)-induced proliferation in epatic stellate cells (HSCs), primary effector cells in liver fibrosis, is mediated by the PTPs SHP-2 and SHP-1: SHP-2 acts as positive regulator of PDGF-dependent signalling, whereas SHP-1 is a negative regulator and lead to the dephosphorylation of the PDGF-receptor. An altered activity and/or expression of these two PTPs causes an inhibited cell proliferation of HSCs, thus they may be a target for new antifibrotic therapies for patients with liver fibrosis. The three pathological models analyzed in this work highlight the key role of PTPs in signalling pathways; they are not to be trivially dismissed as negative regulators because they can organize cellular responses to different stimulations. Furher studies on PTPs-induced signalling regulation may identify new pharmacological therapies
La fosforilazione tirosinica di proteine degli organismi eucarioti è il meccanismo chiave di trasduzione del segnale indotto da stimoli ambientali a livello cellulare, e si esplica in eventi di proliferazione, differenziazione, morte cellulare e sopravvivenza. Questo processo è mediato dall’azione concertata di protein tirosin chinasi (PTKs), che trasferiscono un gruppo fosfato alle proteine bersaglio, e protein tirosin fosfatasi (PTPs), che lo rimuovono; entrambe le classi di questi enzimi possono prender parte ad eventi attivatori e inibitori del signalling. La fosforilazione in tirosina determina il coinvolgimento anche del dominio SH2 (Src-homology 2 domain), presente in numerose proteine con svariate funzioni, che coordina l’interazione proteina-proteina proprio grazie ai residui tirosinici fosforilati, prendendo quindi parte alla modulazione del segnale di trasduzione. Il sistema formato da PTKs, PTPs e dominio SH2 è noto come “writer” (PTK), “eraser” (PTP) e “reader”(SH2) tollkit, denominazione che vuole sottolineare come la relazione e la combinazione di questi tre elementi riesca a determinare una fine e complessa regolazione a livello di signalling. Questo lavoro di tesi intende esaminare il coinvolgimento della fosforilazione tirosinica nel mediare le risposte cellulari a diversi stimoli extracellulari e valutare quali sono i fattori responsabili della sua alterazione in determinate situazioni patologiche ed in particolare il ruolo delle PTPs non recettoriali in tre specifiche patologie. In Policitemia Vera (PV) e Trombocitemia Essenziale (ET), disordini mieloproliferativi Philadelphia-negative (Ph-MPDs), i nostri dati dimostrano che la mancata fosforilazione del sito inibitorio, Y527, della tirosin chinasi Src in piastrine non stimolate è dovuto alla costitutiva attivazione di SHP-2, una PTP non recettoriale, determinando una forma pre-attivata di Src. Questa forma di Src è implicata nella ipersensibilizzazione piastrinica ed è responsabile, almeno parzialmente, delle funzionalità anormali delle piastrine di PV e ET. Nella Trombocitopenia Indotta da Eparina (HIT), reazione autoimmune che porta all’attivazione del recettore FcγRIIA piastrinico, pur non avendo evidenziato una correlazione tra il polimorfismo del recettore e HIT, noi abbiamo indagato il diverso stato di fosforilazione della sequenza ITAM di FcγRIIA e conseguente risposta piastrinica in relazione al tipo di ligando. Stimolando infatti le piastrine di donatori con l’anticorpo monoclonale IV.3, noto per essere un inibitore di FcγRIIA, e con delle IgG complessate, non abbiamo evidenziato fosforilazione dell’ITAM del recettore nè aggregazione, anche se le piastrine vanno incontro ad un cambiamento di forma. In presenza però dell’inibitore di PTP1B, la fosfatasi più abbondante in questo tipo di cellule, gli stessi stimoli portano alla fosforilazione di ITAM e le piastrine vanno incontro ad aggregazione. Questo meccanismo potrebbe essere implicato nel ruolo svolto dalle piastrine nella clereance dei complessi contenenti IgG presenti in circolo. Infine noi abbiamo dimostrato per la prima volta che la risposta proliferativa dopo stimolazione di PDGF (platelet-derived growth factor) delle cellule stellate epatiche (HSCs), principali cellule coinvolte nella fibrosi epatica, è mediata dalle fosfatasi SHP-2 e SHP-1: SHP-2 partecipa come trasduttore positivo del segnale mediato da PDGF, mentre SHP-1 ha effetto negativo sul segnale mediato da PDGF catalizzando la defosforilazione del recettore di PDGF. L’alterazione della loro attività e/o espressione porta ad una inibizione della proliferazione cellulare di HSCs, perciò entrambe le fosfatasi si propongono come possibili bersagli di potenziali farmaci antifibrotici. In tutti i modelli patologici studiati, è chiaro che le PTPs rivestono un ruolo fondamentale nel signaling cellulare; la loro presenza non solo non ha significato esclusivamente negativo ma è necessaria per organizzare la risposta a diversi tipi di stimolo. Lo studio della regolazione del signaling indotto dalle PTPs potrebbe quindi aprire strade alternative per individuare nuove terapie farmacologiche