Expression of the repressor element-1 silencing transcription factor (REST) is regulated by IGF-I and PKC in human neuroblastoma cells

The repressor element 1-silencing transcription factor (REST) was first identified as a protein that binds to a 21-bp DNA sequence element (known as repressor element 1 (RE1)) resulting in transcriptional repression of the neural-specific genes [Chong et al., 1995; Schoenherr and Anderson, 1995]. The original proposed role for REST was that of a factor responsible for restricting neuronal gene expression to the nervous system by silencing expression of these genes in non-neuronal cells. Although it was initially thought to repress neuronal genes in non-neuronal cells, the role of REST is complex and tissue dependent. In this study I investigated any role played by REST in the induction and patterning of differentiation of SH-SY5Y human neuroblastoma cells exposed to IGF-I. and phorbol 12- myristate 13-acetate (PMA) To down-regulate REST expression we developed an antisense (AS) strategy based on the use of phosphorothioate oligonucleotides (ODNs). In order to evaluate REST mRNA levels, we developed a real-time PCR technique and REST protein levels were evaluated by western blotting. Results showed that nuclear REST is increased in SH-SY5Y neuroblastoma cells cultured in SFM and exposed to IGF-I for 2-days and it then declines in 5-day-treated cells concomitant with a progressive neurite extension. Also the phorbol ester PMA was able to increase nuclear REST levels after 3-days treatment concomitant to neuronal differentiation of neuroblastoma cells, whereas, at later stages, it is down-regulated. Supporting these data, the exposure to PKC inhibitors (GF10923X and Gö6976) and PMA (16nM) reverted the effects observed with PMA alone. REST levels were related to morphological differentiation, expression of growth coneassociated protein 43 (GAP-43; a gene not regulated by REST) and of synapsin I and βIII tubulin (genes regulated by REST), proteins involved in the early stage of neuronal development. We observed that differentiation of SH-SY5Y cells by IGF-I and PMA was accompanied by a significant increase of these neuronal markers, an effect that was concomitant with REST decrease. In order to relate the decreased REST expression with a progressive neurite extension, I investigated any possible involvement of the ubiquitin–proteasome system (UPS), a multienzymatic pathway which degrades polyubiquinated soluble cytoplasmic proteins [Pickart and Cohen, 2004]. For this purpose, SH-SY5Y cells are concomitantly exposed to PMA and the proteasome inhibitor MG132. In SH-SY5Y exposed to PMA and MG 132, we observed an inverse pattern of expression of synapsin I and β- tubulin III, two neuronal differentiation markers regulated by REST. Their cytoplasmic levels are reduced when compared to cells exposed to PMA alone, as a consequence of the increase of REST expression by proteasome inhibitor. The majority of proteasome substrates identified to date are marked for degradation by polyubiquitinylation; however, exceptions to this principle, are well documented [Hoyt and Coffino, 2004]. Interestingly, REST degradation seems to be completely ubiquitin-independent. The expression pattern of REST could be consistent with the theory that, during early neuronal differentiation induced by IGF-I and PKC, it may help to repress the expression of several genes not yet required by the differentiation program and then it declines later. Interestingly, the observation that REST expression is progressively reduced in parallel with cell proliferation seems to indicate that the role of this transcription factor could also be related to cell survival or to counteract apotosis events [Lawinger et al., 2000] although, as shown by AS-ODN experiments, it does not seem to be directly involved in cell proliferation. Therefore, the decline of REST expression is a comparatively later event during maturation of neuroroblasts in vitro. Thus, we propose that REST is regulated by growth factors, like IGF-I, and PKC activators in a time-dependent manner: it is elevated during early steps of neural induction and could contribute to down-regulate genes not yet required by the differentiation program while it declines later for the acquisition of neural phenotypes, concomitantly with a progressive neurite extension. This later decline is regulated by the proteasome system activation in an ubiquitin-indipendent way and adds more evidences to the hypothesis that REST down-regulation contributes to differentiation and arrest of proliferation of neuroblastoma cells. Finally, the glycosylation pattern of the REST protein was analysed, moving from the observation that the molecular weight calculated on REST sequence is about 116 kDa but using western blotting this transcription factor appears to have distinct apparent molecular weight (see Table 1.1): this difference could be explained by post-translational modifications of the proteins, like glycosylation. In fact recently, several studies underlined the importance of O-glycosylation in modulating transcriptional silencing, protein phosphorylation, protein degradation by proteasome and protein–protein interactions [Julenius et al., 2005; Zachara and Hart, 2006]. Deglycosilating analysis showed that REST protein in SH-SY5Y and HEK293 cells is Oglycosylated and not N-glycosylated. Moreover, using several combination of deglycosilating enzymes it is possible to hypothesize the presence of Gal-β(1-3)-GalNAc residues on the endogenous REST, while β(1-4)-linked galactose residues may be present on recombinant REST protein expressed in HEK293 cells. However, the O-glycosylation process produces an immense multiplicity of chemical structures and monosaccharides must be sequentially hydrolyzed by a series of exoglycosidase. Further experiments are needed to characterize all the post-translational modification of the transcription factor REST.

Genomic and non genomic effects of elevated concentration of anabolic steroids in human neuronal cells

Nandrolone and other anabolic androgenic steroids (AAS) at elevated concentration can alter the expression and function of neurotransmitter systems and contribute to neuronal cell death. This effect can explain the behavioural changes, drug dependence and neuro degeneration observed in steroid abuser. Nandrolone treatment (10-8M–10-5M) caused a time- and concentration-dependent downregulation of mu opioid receptor (MOPr) transcripts in SH-SY5Y human neuroblastoma cells. This effect was prevented by the androgen receptor (AR) antagonist hydroxyflutamide. Receptor binding assays confirmed a decrease in MOPr of approximately 40% in nandrolonetreated cells. Treatment with actinomycin D (10-5M), a transcription inhibitor, revealed that nandrolone may regulate MOPr mRNA stability. In SH-SY5Y cells transfected with a human MOPr luciferase promoter/reporter construct, nandrolone did not alter the rate of gene transcription. These results suggest that nandrolone may regulate MOPr expression through post-transcriptional mechanisms requiring the AR. Cito-toxicity assays demonstrated a time- and concentration dependent decrease of cells viability in SH-SY5Y cells exposed to steroids (10-6M–10-4M). This toxic effects is independent of activation of AR and sigma-2 receptor. An increased of caspase-3 activity was observed in cells treated with Nandrolone 10-6M for 48h. Collectively, these data support the existence of two cellular mechanisms that might explain the neurological syndromes observed in steroids abuser.

A step forwards in immunomodulation and immunetolerance knowledge. HLA-G expression in co-cultures of peripheral blood mononuclear cells and stem cells after in vitro NGAL stimulation

NGAL (Neutrophil Gelatinase-associated Lipocalin ) is a protein of lipocalin superfamily. Recent literature focused on its biomarkers function in several pathological condition (acute and chronic kidney damage, autoimmune disease, malignancy). NGAL biological role is not well elucidated. Several are the demonstration of its bacteriostatic role. Recent papers have indeed highlight NGAL role in NFkB modulation. The aim of this study is to understand whether NGAL may exert a role in the activation (modulation) of T cell response through the regulation of HLA-G complex, a mediator of tolerance. From 8 healthy donors we obtained peripheral blood mononuclear cells (PBMCs) and we isolated by centrifugation on a Ficoll gradient. Cells were then treated with four concentrations of NGAL (40-320 ng/ml) with or without iron. We performed flow cytometry analysis and ELISA test. NGAL increased the HLA-G expression on CD4+ T cells, with an increasing corresponding to the dose. Iron effect is not of unique interpretation. NGAL adiction affects regulatory T cells increasing in vitro expansion of CD4+ CD25+ FoxP3+ cells. Neutralizing antibody against NGAL decreased HLA-G expression and reduced significantly CD4+ CD25+ FoxP3+ cells percentage. In conclusion, we provided in vitro evidence of NGAL involvement in cellular immunity. The potential role of NGAL as an immunomodulatory molecule has been evaluated: it has been shown that NGAL plays a pivotal role in the induction of immune tolerance up regulating HLA-G and T regulatory cells expression in healthy donors. As potential future scenario we highlight the in vivo role of NGAL in immunology and immunomodulation, and its possible relationship with immunosuppressive therapy efficacy, tolerance induction in transplant patients, and/or in other immunological disorders.

Airway Basal Cell Vascular Endothelial Growth Factor-mediated Cross-Talk Regulates Endothelial Cell Dependent Growth Support of Human Airway Basal Cells

The human airway epithelium is a pseudostratified heterogenous layer comprised of cili-ated, secretory, intermediate and basal cells. As the stem/progenitor population of the airway epi-thelium, airway basal cells differentiate into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. Transcriptome analysis of airway basal cells revealed high expression of vascular endothelial growth factor A (VEGFA), a gene not typically associated with the function of this cell type. Using cultures of primary human airway basal cells, we demonstrate that basal cells express all of the 3 major isoforms of VEGFA (121, 165 and 189) but lack functional expression of the classical VEGFA receptors VEGFR1 and VEGFR2. The VEGFA is actively secreted by basal cells and while it appears to have no direct autocrine function on basal cell growth and proliferation, it functions in a paracrine manner to activate MAPK signaling cascades in endothelium via VEGFR2 dependent signaling pathways. Using a cytokine- and serum-free co-culture system of primary human airway basal cells and human endothelial cells revealed that basal cell secreted VEGFA activated endothelium to ex-press mediators that, in turn, stimulate and support basal cell proliferation and growth. These data demonstrate novel VEGFA mediated cross-talk between airway basal cells and endothe-lium, the purpose of which is to modulate endothelial activation and in turn stimulate and sustain basal cell growth.

Controllo molecolare del differenziamento osteoblestico per applicazioni nel campo della rigenerazione del tessuto osseo

The aim of the study was to identify expression signatures unique for specific stages of osteoblast differentiation in order to improve our knowledge of the molecular mechanisms underlying bone repair and regeneration. We performed a microarray analysis on the whole transcriptome of human mesenchymal stem cells (hMSCs) obtained from the femoral canal of patients undergoing hip replacement. By defining different time-points within the differentiation and mineralization phases of hMSCs, temporal gene expression changes were visualised. Importantly, the gene expression of adherent bone marrow mononuclear cells, being the undifferentiated progenitors of bone cells, was used as reference. In addition, only the cultures able to form mineral nodules at the final time-point were considered for the gene expression analyses. To obtain the genes of our interest, we only focused on genes: i) whose expression was significantly upregulated; ii) which are involved in pathways or biological processes relevant to proliferation, differentiation and functions of bone cells; iii) which changed considerably during the different steps of differentiation and/or mineralization. Among the 213 genes identified as differentially expressed by microarray analysis, we selected 65 molecular markers related to specific steps of osteogenic differentiation. These markers are grouped into various gene clusters according to their involvement in processes which play a key role in bone cell biology such as angiogenesis, ossification, cell communication, development and in pathways like TGF beta and Wnt signaling pathways. Taken together, these results allow us to monitor hMSC cultures and to distinguish between different stages of differentiation and mineralization. The signatures represent a useful tool to analyse a broad spectrum of functions of hMSCs cultured on scaffolds, especially when the constructs are conceived for releasing growth factors or other signals to promote bone regeneration. Morover, this work will enhance our understanding of bone development and will enable us to recognize molecular defects that compromise normal bone function as occurs in pathological conditions.

Metodi biomolecolari per il follow up dei pazienti HIV positivi

As proviral human immunodeficiency virus type 1 (HIV-1) DNA can replenish and revive viral infection upon attivation, its analysis, in addition to RNA viral load, could be considered a useful marker during the follow-up of infected individuals, to evaluate reservoir status, especially in HAART-treated patients when RNA viral load is undetectable by current techniques and the antiretroviral efficacy of new, more potent therapeutic regimens. Standardized methods for the measurement of the two most significant forms of proviral DNA, total and non-integrated, are currently lacking, despite the widespread of molecular biology techniques. In this study, total and 2-LTR HIV-1 DNA proviral load, in addition to RNA viral load, CD4 cell count and serological parameters, were determined by quantitative analysis in peripheral blood mononuclear cells (PBMC) in naïve or subsequently HAART-treated patients with acute HIV-1 infection in order to establish the role of these two DNA proviral forms in the course of HIV infection. The study demonstrated that HAART-treated individuals show a significant decrease in both total and 2-LTR circular HIV-1 DNA proviral load compared with naïve patients: these findings confirm that HIV-1 reservoir decay correlates with therapeutic effectiveness. The persistence of small amounts of 2-LTR HIV-1 DNA form, which is considered to be a molecular determinant of infectivity, in PBMC from some patients demonstrates that a small rate of replication is retained even when HAART is substantially effective: HAART could not eradicate completely the infection because HIV is able to replicate at low levels. Plasma-based viral RNA assays may fail to demonstrate the full extent of viral activity. In conclusion, the availability of a new standardized assay to determine DNA proviral load will be important in assessing the true extent of virological suppression suggesting that its quantification may be an important parameter in monitoring HIV infection.

Computational modelling of human stem cell-derived cardiomyocytes - Texture descriptors for biological image processing

This thesis investigates two distinct research topics. The main topic (Part I) is the computational modelling of cardiomyocytes derived from human stem cells, both embryonic (hESC-CM) and induced-pluripotent (hiPSC-CM). The aim of this research line lies in developing models of the electrophysiology of hESC-CM and hiPSC-CM in order to integrate the available experimental data and getting in-silico models to be used for studying/making new hypotheses/planning experiments on aspects not fully understood yet, such as the maturation process, the functionality of the Ca2+ hangling or why the hESC-CM/hiPSC-CM action potentials (APs) show some differences with respect to APs from adult cardiomyocytes. Chapter I.1 introduces the main concepts about hESC-CMs/hiPSC-CMs, the cardiac AP, and computational modelling. Chapter I.2 presents the hESC-CM AP model, able to simulate the maturation process through two developmental stages, Early and Late, based on experimental and literature data. Chapter I.3 describes the hiPSC-CM AP model, able to simulate the ventricular-like and atrial-like phenotypes. This model was used to assess which currents are responsible for the differences between the ventricular-like AP and the adult ventricular AP. The secondary topic (Part II) consists in the study of texture descriptors for biological image processing. Chapter II.1 provides an overview on important texture descriptors such as Local Binary Pattern or Local Phase Quantization. Moreover the non-binary coding and the multi-threshold approach are here introduced. Chapter II.2 shows that the non-binary coding and the multi-threshold approach improve the classification performance of cellular/sub-cellular part images, taken from six datasets. Chapter II.3 describes the case study of the classification of indirect immunofluorescence images of HEp2 cells, used for the antinuclear antibody clinical test. Finally the general conclusions are reported.

Contribution of vascular resident mesenchymal stromal cells to abdominal aortic aneurysm pathogenesis: increased MMP-9 expression and ineffective immunomodulation

Background. Ageing and inflammation are critical for the occurrence of aortic diseases. Extensive inflammatory infiltrate and excessive ECM proteloysis, mediated by MMPs, are typical features of abdominal aortic aneurysm (AAA). Mesenchymal Stromal Cells (MSCs) have been detected within the vascular wall and represent attractive candidates for regenerative medicine, in virtue of mesodermal lineage differentiation and immunomodulatory activity. Meanwhile, many works have underlined an impaired MSC behaviour under pathological conditions. This study was aimed to define a potential role of vascular MSCs to AAA development. Methods. Aortic tissues were collected from AAA patients and healthy donors. Our analysis was organized on three levels: 1) histology of AAA wall; 2) detection of MSCs and evaluation of MMP-9 expression on AAA tissue; 3) MSC isolation from AAA wall and characterization for mesenchymal/stemness markers, MMP-2, MMP-9, TIMP-1, TIMP-2 and EMMPRIN. AAA-MSCs were tested for immunomodulation, when cultured together with activated peripheral blood mononuclear cells (PBMCs). In addition, a co-colture of both healthy and AAA MSCs was assessed and afterwards MMP-2/9 mRNA levels were analyzed. Results. AAA-MSCs showed basic mesenchymal properties: fibroblastic shape, MSC antigens, stemness genes. MMP-9 mRNA, protein and enzymatic activity were significantly increased in AAA-MSCs. Moreover, AAA-MSCs displayed a weak immunosuppressive activity, as shown by PBMC ongoing along cell cycle. MMP-9 was shown to be modulated at the transcriptional level through the direct contact as well as the paracrine action of healthy MSCs. Discussion. Vascular injury did not affect the MSC basic phenotype, but altered their function, a increased MMP-9 expression and ineffective immunmodulation. These data suggest that vascular MSCs can contribute to aortic disease. In this view, the study of key processes to restore MSC immunomodulation could be relevant to find a pharmacological approach for monitoring the aneurysm progression.

Unbalanced R-loops and micronuclei induced by DNA topoisomerase I poisons in cancer cells

Topoisomerase I (Top1) poisons are among the most clinically-effective drugs used for colon, ovary and lung cancers. Unpublished data from our lab have recently revealed that the structurally-unrelated Top1 poisons, Camptothecin (CPT) and Indimitecan (LMP776), induce the formation of micronuclei (MNi) in human cancer cells. In addition, MNi trigger an innate immune gene response by stimulating the cGAS/STING pathway. As the mechanisms of MNi formation are not fully determined, our aim is here to establish how MNi form after Top1 poisoning. Using immunofluorescence assays and EdU labelling of nascent DNAs, our results show that, after 24 hours of recovery, a short treatment with sub-cytotoxic doses of Top1 poisons induces the formation of MNi that do not contain newly synthetized (EdU+) DNA. We also saw that Top1 poisons delay replication machinery reducing EdU incorporation and produce significant levels of the damage markers γH2AX and p53BP1 in S-phase cells but not in G1 and G2/M cells. The results also show that MNi formation is dependent on R-loops, as RNaseH1 overexpression markedly reduces Top1 induced MNi. Genome-wide mapping of R-loops by DRIP-seq technique revealed that R-loop levels are both decreased and increased by CPT. In particular, increased R-loops are mainly found at active genes and always overlapped with Top1cc sites. We also found that increased R-loops overlap with lamina-associated chromatin domains while decreased R-loops correlate with replication origin sites. Overall, our data are consistent with the formation of MNi due to R-loop increase and under-replication at specific regions caused by Top1 poisons. These results will eventually help in developing new strategies for effective personalized interventions by using Top1-targeted compounds as immuno-modulators in cancer patients.