Trasduzione del segnale e poliamine nell'apoptosi di cellule cardiache

Introduction: Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy and heart failure, thus representing a potential therapeutic target. Apoptosis of cardiac cells can be induced experimentally by several stimuli including hypoxia, serum withdrawal or combination of both. Several lines of research suggest that neurohormonal mechanisms play a central role in the progression of heart failure. In particular, excessive activation of the sympathetic nervous system or the renin-angiotensin-aldosterone system is known to have deleterious effects on the heart. Recent studies report that norepinephrine (NE), the primary transmitter of sympathetic nervous system, and aldosterone (ALD), which is actively produced in failing human heart, are able to induce apoptosis of rat cardiomyocytes. Polyamines are biogenic amines involved in many cellular processes, including apoptosis. Actually it appears that these molecules can act as promoting, modulating or protective agents in apoptosis depending on apoptotic stimulus and cellular model. We have studied the involvement of polyamines in the apoptosis of cardiac cells induced in a model of simulated ischemia and following treatment with NE or ALD. Methods: H9c2 cardiomyoblasts were exposed to a condition of simulated ischemia, consisting of hypoxia plus serum deprivation. Cardiomyocyte cultures were prepared from 1-3 day-old neonatal Wistar rat hearts. Polyamine depletion was obtained by culturing the cells in the presence of α-difluoromethylornithine (DFMO). Polyamines were separated and quantified in acidic cellular extracts by HPLC after derivatization with dansyl chloride. Caspase activity was measured by the cleavage of the fluorogenic peptide substrate. Ornithine decarboxylase (ODC) activity was measured by estimation of the release of 14C-CO2 from 14C-ornithine. DNA fragmentation was visualized by the method of terminal transferase-mediated dUTP nick end-labeling (TUNEL), and DNA laddering on agarose gel electophoresis. Cytochrome c was detected by immunoflorescent staining. Activation of signal transduction pathways was investigated by western blotting. Results: The results indicate that simulated ischemia, NE and ALD cause an early induction of the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, followed by a later increase of caspase activity, a family of proteases that execute the death program and induce cell death. This effect was prevented in the presence of DFMO, an irreversible inhibitor of ODC, thus suggesting that polyamines are involved in the execution of the death program activated by these stimuli. In H9c2 cells DFMO inhibits several molecular events related to apoptosis that follow simulated ischemia, such as the release of cytochrome c from mitochondria, down-regulation of Bcl-xL, and DNA fragmentation. The anti-apoptotic protein survivin is down-regulated after ALD or NE treatement and polyamine depletion obtained by DFMO partially opposes survivin decrease. Moreover, a study of key signal transduction pathways governing cell death and survival, revealed an involvement of AMP activated protein kinase (AMPK) and AKT kinase, in the modulation by polyamines of the response of cardiomyocytes to NE. In fact polyamine depleted cells show an altered pattern of AMPK and AKT activation that may contrast apoptosis and appears to result from a differential effect on the specific phosphatases that dephosphorylate and switch off these signaling proteins. Conclusions: These results indicate that polyamines are involved in the execution of the death program activated in cardiac cells by heart failure-related stimuli, like ischemia, ALD and NE, and suggest that their apoptosis facilitating action is mediated by a network of specific phosphatases and kinases.

Studio del pathway della Displasia Ectodermica Anidrotica (EDA)

Anhidrotic Ectodermal Dysplasia (EDA), is the most frequent form among Ectodermal Dysplasias, hereditary genetic disorders causing ectodermal appendages defective development. Indeed, EDA is characterized by defective formation of hair follicles, sweat glands and teeth both in human patients and animals. EDA, the gene mutated in Anhidrotic Ectodermal Dysplasia, encodes Ectodysplasin, a TNF family member that activates NF-kB mediated transcription. This disease can occur with mutations in other EDA-NF-kB pathway members, as EDA receptor, EDAR and its adapter, EDARADD. Moreover, mutations in TRAF6, NEMO, IKB and NF-kBs genes are responsible for Immunodeficiency associated EDA (EDA-ID). Several molecules, as SHH, WNT/DKK, BMP and LTβ, have already been reported to be EDA pathway regulators or effectors although the knowledge of the full spectrum of EDA targets remains incomplete. During the first part of the research project a gene expression analysis was performed in primary keratinocytes from Wild-type and Tabby (EDA model mouse) mice to identify novel EDA target genes. Earlier expression profiling at various developmental time points in Tabby and Wild-type mouse skin reported genes differentially expressed in the two samples and, to increase the resolution to find genes whose expression may be restricted to epidermal cells, the study was extended to primary keratinocyte cultures established from E19 Wild-type and Tabby skin. Using microarrays bearing 44,000 gene probes, we found 385 “preliminary candidate” genes whose expression was significantly affected by Eda defect. By comparing expression profiles to those from Eda-A1 (where Eda-A1 is highly expressed) transgenic skin, we restricted the list to 38 “candidate EDA targets”, 14 of which were already known to be expressed in hair follicles or epidermis. This work confirmed expression changes for 3 selected genes, Tbx1, Bmp7, and Jag1, both in primary keratinocytes and in Wild-type and Tabby whole skin, by Q-PCR and Western blotting analyses. Thus, this study detected novel candidate pathways downstream of EDA. In the second part of the research project, plasmid constructs were produced and analyzed to create a transgenic mouse model for Immunodeficiency associated EDA disease (XL-EDA-ID). In particular, plasmids containing mouse Wild-type and mutated Nemo cDNA under K-17 epidermis-specific promoter control and a Flag tag, were prepared, on the way to confine transgene expression to mice epidermis and to determine EDA phenotype without immunodeficiency for a comparison to Tabby model phenotype. EDA-ID mutations reported in patients and selected for this study are: C417R (C409R in mouse), causing Zinc Finger protein domain destabilization and A288G (A282G in mouse) affecting oligomerization of the protein. Moreover, the ex-novo mutation, ZnF, C-terminal Zinc Finger domain deletion, was tested. Thus, the constructs were analyzed by transient transfection, Western blotting and luciferase assays techniques, detecting Nemo Wild-type and mutant protein products and residue NF-kB activity in presence of mutants, after TNF stimulation. In particular, MEF_Nemo-/- cell line was used to monitor NF-kB activity without endogenous Nemo gene. Results show reduced NF-kB activity in presence of mutated Nemo forms compared to Wild-type: 81% for A282G (A288G in human); 24% for C409R (C417R in human); 15% for ZnF. C409R mutation (C417R in human), reported in 6 EDA-ID human patients, was selected to prepare transgenic model mouse. Mice (white, FVP) born following K17-promoter-Flag-Nemo_C409R plasmid region pronuclear injection, were analyzed for the transgene presence in the genotype and a preliminar examination of their phenotype was performed. In particular, one mouse showed considerable coat defects if compared to Wild-type mice. This preliminar analysis suggests a possible influence of Nemo mutant over-expression in epidermis without immunodeficiency. Still, more microscopic studies to analyze hair subtypes, Guard, Awl and Zigzag (usually alterated inTabby mouse model), Immunohistochemistry experiments to detect epidermis restricted Nemo expression and sweat glands analysis, will follow. This and other transgene positive mice will be crossed with black mice C57BL6 to obtain at least two indipendent agouti lines to analyze. Theses mice will be used in EDA target genes detection through microarrays. Following, plasmid constructs containing other Nemo mutant forms (A282G and ZnF) might be studied by the same experimental approaches to prepare more transgenic model mice to compare to Nemo_C409R and Tabby mouse models.

Caratterizzazione e ruolo di PKCε e PKCδ in modelli di differenziamento megacariocitario normale e patologico

La PKCε e la PKCδ, chinasi ubiquitariamente distribuite e ad azione pleiotropica, sono implicate del differenziamento, sopravvivenza e proliferazione cellulare. Esse sono coinvolte nel processo differenziativo delle cellule staminali ematopoietiche e in fenomeni patologici associati al compartimento sanguigno. In questa tesi sono presentati i risultati riguardanti lo studio in vitro del ruolo di PKCε e PKCδ nel contesto del differenziamento megacariocitario, in particolare si caratterizza l’espressione e la funzione di queste chinasi nel modello umano e nel modello murino di Megacariocitopoiesi, normale e patologica. Confrontando le cinetiche dei due modelli presi in analisi nello studio è stato possibile osservare come in entrambi PKCε e PKCδ dimostrino avere una chiara e specifica modulazione nel progredire del processo differenziativo. Questi dati, se confrontati, permettono di affermare che PKCε e PKCδ presentano un pattern di espressione opposto e, nel modello umano rispetto a quello murino, reciproco: nell’uomo i livelli di PKCε devono essere down-modulati, mentre nel topo, al contrario, i livelli della chinasi risultano up-modulati durante lo stesso processo. Analogamente, le CD34+ in differenziazione presentano una costante e maggiore espressione di PKCδ durante la maturazione MK, mentre nel modello murino tale proteina risulta down-modulata nella fase più tardiva di formazione della piastrina. Le chinasi mostrano in oltre di agire, nei due modelli, attraverso pathways distinti e cioè RhoA nel topo e Bcl-xL nell’uomo. È stato inoltre verificato che l’aberrante differenziamento MK osservato nella mielofibrosi primaria (PMF), è associato a difetti di espressione di PKCε e di Bcl-xL e che una forzata down-modulazione di PKCε porta ad un ripristino di un normale livello di espressione di Bcl-xL così come della popolazione di megacariociti formanti propiastrine. I dati ottenuti indicano quindi che PKCε e PKCδ svolgono un ruolo importante nel corretto differenziamento MK e che PKCε potrebbe essere un potenziale nuovo target terapeutico nelle PMF.