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.

Bioverkapselung lebender Bakterien (Escherichia coli) mit Poly-(Silicat) nach Transformation mit dem Silicatein-alpha-Gen

Die Bioverkapselung ist eine faszinierende Methode, um biologische Materialien einschließlich Zellen in Siliziumdioxid, Metalloxiden oder hybriden Sol-Gel-Polymeren zu immobilisieren. Bisher wurde nur die Sol-Gel-Vorläufertechnologie genutzt, um Bakterien- oder Hefezellen in Siliziumdioxid zu immobilisieren. Hierfür wurden verschiedene Reagenzien als wässrige Vorläufer getestet, um poly(Silicate) auf Biomolekülen (Bhatia et al., 2000) oder Zellen (Liu und Chen 1999; Coradin und Livage, 2007) zu bilden. Einer der erfolgreichsten bisherigen Methoden verwendet eine Mischung aus Silicaten und kolloidalem Silica. Diese initialen Vorläufer werden durch die Zugabe von Salzsäure neutralisiert, was die Gelbildung fortschreiten lässt und die Verkapselung von Bakterien in einem Silica-Netzwerk zur Folge hat (Nassif et al., 2003). Mit der Entdeckung von Silicatein, einem Enzym, das aus Demospongien isoliert wurde und die Bildung von poly(Silicat) katalysiert, wurde es möglich, poly(Silicat) unter physiologischen Bedingungen zu synthetisieren. Silicatein wurde rekombinant in E. coli hergestellt und ist in der Lage, bei Raumtemperatur, neutralem pH-Wert und in wässrigen Puffersystemen aus Siliziumalkoxiden poly(Silicat) zu bilden (Krasko et al., 2000; Müller et al., 2007b; Zhou et al., 1999). In vivo katalysiert Silicatein die Synthese der Silicathülle der Schwamm-Spiculae (Skelettelemente; Müller et al., 2005b; Müller et al., 2007a; Müller et al., 2007b; Schröder et al., 2007a). Dieses Biosilica wurde in Form von Silica-Nanospheren mit Durchmessern zwischen 100 nm und 250 nm organisiert vorgefunden (Pisera 2003; Tahir et al., 2005). Mit dieser Arbeit konnte gezeigt werden, dass Escherichia coli erfolgreich mit dem Silicatein-Gen transformiert werden kann. Das Level der Proteinexpression kann in Anwesenheit von Isopropyl-β-D-thiogalaktopyranosid (IPTG) effizient erhöht werden, indem man die Bakterienzellen gleichzeitig mit Kieselsäure inkubiert. Dieser Effekt konnte sowohl auf Ebene der Synthese des rekombinanten Proteins durch Western Blot als auch durch Immunfluoreszenzmikroskopie nachgewiesen werden. Das heterolog produzierte Silicatein besitzt enzymatische Aktivität und kann die Polymerisation von Kieselsäure katalysieren. Dies konnte sowohl durch Färbung mit Rhodamin123, als auch durch Reaktion der nicht polymerisierten, freien Kieselsäure mit dem ß-Silicomolybdato-Farbsystem (Silicomolybdänblau) nachgewiesen werden. Elektronenmikroskopische Untersuchungen zeigten, dass nur die silicateinexprimierenden Bakterien während des Wachstums in Anwesenheit von Kieselsäure eine viskose Hülle um Zelle herum bilden. Ebenfalls konnte gezeigt werden, dass Silicatein-α aus Suberites domuncula nach Transformation in E. coli an die Zelloberfläche dieser Zellen transportiert wurde und dort seine enzymatische Funktion beibehielt. Die Silicathülle wurde mittels Raster-Elektronenmikroskopie (REM) analysiert. Die Bakterien, die Silicatein exprimierten und poly(Silicat) an ihrer Oberfläche synthetisierten, zeigten die gleichen Wachstumsraten wie die Bakterien, die das Gen nicht enthielten. Schlussfolgernd lässt sich sagen, dass die silicateinvermittelte Verkapselung von Bakterien mit poly(Silicat) die Bandbreite der Anwendung von Bakterien für die Produktion von rekombinanten Proteinen verbessern, erweitern und optimieren könnte.

Purification and Preliminary Characterization of the TM0727 Protein from Thermotoga maritima

The TM0727 gene of Thermotoga maritima is responsible for encoding what has been reported to be a modulator of DNA gyrase (pmbA). Although the function of pmbA is still unknown, it is believedto be involved in cell division, carbon storage regulation, and the synthesis of the antibiotic peptide microcin B17. It is suggested that it serves together with tldD, a known zinc dependent protease, tomodulate DNA gyrase. TM0727 is believed to be a zinc dependent protease that binds zinc in the central active site of the molecule, located between two equivalent monomeric units. However, thecrystal structure determined by Wilson et al. (2005) did not contain zinc. It therefore remains to be seen if TM0727 requires zinc for activity, or regulation, and if the protein is indeed a protease. To begin studying this protein, the gene was expressed in BL21(DE3) pLysS cells and the induction time was optimized. Using affinity and ion exchange chromatography, the protein has been successfully purified. The purification procedure can be replicated to obtain sufficient protein for characterization. Purification results show that the protein loses stability after 24 hours and remains stable under an imidazole-free lysis workup. Preliminary characterization of TM0727 has focused on understanding the protein’s structuralproperties through tryptophan fluorescence anisotropy measurements. The four tryptophan residues located within the TM0727 dimer fluoresce at different maximum wavelengths and with differentintensities upon excitation with 295nm light. These emission properties are highly sensitive to the environment (solvent, surrounding residues) of each tryptophan residue. The low number oftryptophans allows for a specific monitoring of the protein’s structure as it denatures. As more denaturant is added to the protein, its tryptophan environments have clearly altered. This is indicative of unfolding and increased solvent exposure of the protein. This unfolding has been confirmed with the addition of a fluorescent quencher. Additionally, fluorescence anisotropy measurements have been carried out on the protein to gain a preliminary understanding of the rotational dynamics of the tryptophan residues. These experiments excite the tryptophan residues within the sample using a polarized light source. Polarized emission is then detected, the degree of which depends on the rotational dynamics and local environment of the tryptophan residues. The protein was denatured and the changes in emission were recorded to detect these structural changes. Results have shown a large change in quaternary structure, consistent with a dimer to monomer transition, occurs at 1.5M Guandidine HCl. There has also been an examination of the crystal structure for the location of a potential active site. The inner cavity of the protein was inspected visually to locate a potential location for a catalytic triad, specifically the amino acids found in the active sites of serine, cyteine, and aspartateproteases. It was found that a potential aspartic protease active site may be located between the Asparate286 and Aspartate287 residues. Further investigation is warranted to test this remotepossibility.