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.

Insulin and TOR pathways regulate cellular and organismal growth through the myc oncogene in Drosophila

A large body of literature documents in both mice and Drosophila the involvement of Insulin pathway in growth regulation, probably due to its role in glucose and lipid import, nutrient storage, and translation of RNAs implicated in ribosome biogenesis (Vanhaesebroeck et al. 2001). Moreover several lines of evidence implicate this pathway as a causal factor in cancer (Sale, 2008; Zeng and Yee 2007; Hursting et al., 2007; Chan et al., 2008). With regards to Myc, studies in cell culture have implied this family of transcription factors as regulators of the cell cycle that are rapidly induced in response to growth factors. Myc is a potent oncogene, rearranged and overexpressed in a wide range of human tumors and necessary during development. Its conditional knock-out in mice results in reduction of body weight due to defect in cell proliferation (Trumpp et al. 2001). Evidence from in vivo studies in Drosophila and mammals suggests a critical function for myc in cell growth regulation (Iritani and Eisenman 1999; Johnston et al. 1999; Kim et al. 2000; de Alboran et al. 2001; Douglas et al. 2001). This role is supported by our analysis of Myc target genes in Drosophila, which include genes involved in RNA binding, processing, ribosome biogenesis and nucleolar function (Orain et al 2003, Bellosta et al., 2005, Hulf et al, 2005). The fact that Insulin signaling and Myc have both been associated with growth control suggests that they may interact with each other. However, genetic evidence suggesting that Insulin signaling regulates Myc in vivo is lacking. In this work we were able to show, for the first time, a direct modulation of dMyc in response to Insulin stimulation/silencing both in vitro and in vivo. Our results suggest that dMyc up-regulation in response to DILPs signaling occurs both at the mRNA and potein level. We believe dMyc protein accumulation after Insulin signaling activation is conditioned to AKT-dependent GSK3β/sgg inactivation. In fact, we were able to demonstate that dMyc protein stabilization through phosphorylation is a conserved feature between Drosophila and vertebrates and requires multiple events. The final phosphorylation step, that results in a non-stable form of dMyc protein, ready to be degraded by the proteasome, is performed by GSK3β/sgg kinase (Sears, 2004). At the same time we demonstrated that CKI family of protein kinase are required to prime dMyc phosphorylation. DILPs and TOR/Nutrient signalings are known to communicate at several levels (Neufeld, 2003). For this reason we further investigated TOR contribution to dMyc-dependent growth regulation. dMyc protein accumulates in S2 cells after aminoacid stimulation, while its mRNA does not seem to be affected upon TORC1 inhibition, suggesting that the Nutrient pathway regulates dMyc mostly post-transcriptionally. In support to this hypothesis, we observed a TORC1-dependent GSK3β/sgg inactivation, further confirming a synergic effect of DILPs and Nutrients on dMyc protein stability. On the other hand, our data show that Rheb but not S6K, both downstream of the TOR kinase, contributes to the dMyc-induced growth of the eye tissue, suggesting that Rheb controls growth independently of S6K.. Moreover, Rheb seems to be able to regulate organ size during development inducing cell death, a mechanism no longer occurring in absence of dmyc. These observations suggest that Rheb might control growth through a new pathway independent of TOR/S6K but still dependent on dMyc. In order to dissect the mechanism of dMyc regulation in response to these events, we analyzed the relative contribution of Rheb, TOR and S6K to dMyc expression, biochemically in S2 cells and in vivo in morphogenetic clones and we further confirmed an interplay between Rheb and Myc that seems to be indipendent from TOR. In this work we clarified the mechanisms that stabilize dMyc protein in vitro and in vivo and we observed for the first time dMyc responsiveness to DILPs and TOR. At the same time, we discovered a new branch of the Nutrient pathway that appears to drive growth through dMyc but indipendently from TOR. We believe our work shed light on the mechanisms cells use to grow or restrain growth in presence/absence of growth promoting cues and for this reason it contributes to understand the physiology of growth control.

Treatment of knee articular cartilage defects: surgical strategies, results and analysis of factors determining the clinical outcome

Articular cartilage lesions, with their inherent limited healing potential, are hard to treat and remain a challenging problem for orthopedic surgeons. Despite the development of several treatment strategies, the real potential of each procedure in terms of clinical benefit and effects on the joint degeneration processes is not clear. Aim of this PhD project was to evaluate the results, both in terms of clinical and imaging improvement, of new promising procedures developed to address the challenging cartilage pathology. Several studies have been followed in parallel and completed over the 3-year PhD, and are reported in detail in the following pages. In particular, the studies have been focused on the evaluation of the treatment indications of a scaffold based autologous chondrocyte implantation procedure, documenting its results for the classic indication of focal traumatic lesions, as well as its use for the treatment of more challenging patients, older, with degenerative lesions, or even as salvage procedure for more advanced stages of articular degeneration. The second field of study involved the analysis of the results obtained treating lesions of the articular surface with a new biomimetic osteochondral scaffold, which showed promise for the treatment of defects where the entire osteochondral unit is involved. Finally, a new minimally invasive procedure based on the use of growth factors derived from autologous platelets has been explored, showing results and underlining indicatios for the treatment of cartilage lesions and different stages of joint degeneration. These studies shed some light on the potential of the evaluated procedures, underlining good results as well as limits, they give some indications on the most appropriate candidates for their application, and document the current knowledge on cartilage treatment procedures suggesting the limitations that need to be addressed by future studies to improve the management of cartilage lesions.