Gotowa bibliografia na temat „Megakaryopoiesis”

Abstract Activin A, BMP2, and BMP4, 3 members of the transforming growth factor-β family, are involved in the regulation of hematopoiesis. Here, we explored the role of these molecules in human megakaryopoiesis using an in vitro serum-free assay. Our results highlight for the first time that, in the absence of thrombopoietin, BMP4 is able to induce CD34+ progenitor differentiation into megakaryocytes through all stages. Although we have previously shown that activin A and BMP2 are involved in erythropoietic commitment, these molecules have no effect on human megakaryopoietic engagement and differentiation. Using signaling pathway-specific inhibitors, we show that BMP4, like thrombopoietin, exerts its effects on human megakaryopoiesis through the JAK/STAT and mTor pathways. Inhibition of the BMP signaling pathway with blocking antibodies, natural soluble inhibitors (FLRG or follistatin), or soluble BMP receptors reveals that thrombopoietin uses the BMP4 pathway to induce megakaryopoiesis, whereas the inverse is not occurring. Finally, we show that thrombopoietin up-regulates the BMP4 autocrine loop in megakaryocytic progenitors by inducing their production of BMP4 and up-regulating BMP receptor expression. In summary, this work indicates that BMP4 plays an important role in the control of human megakaryopoiesis.

Abstract Purpose: To describe the morphological evolution of megakaryocytic dysplasia by developing a systematic classification and evaluate the impact of our classification of dys-megakaryopoiesis on prognosis of persons with MDS. Patients and methods: 423 consecutive patients who had received no prior therapy with MDS diagnosed from January 2000 to April 2014 were enrolled. Follow-up data were available for 371 subjects (88%). Date of last follow-up was December 15, 2014 or date of last contact. Median follow-up was 22 months (range, 1¨C180 months). Subjects with lower-risk MDS fall into Revised International Prognostic scoring systems (IPSS-R) categories of very low-, low-, and intermediate-risk groups and those with higher-risk category into the high- and very high-risk groups. We performed CD41 immune staining and proposed a systematic classification of dys-megakaryopoiesis on bone marrow films: (1) micro-megakaryocytes (<12 µm); (2) micro-megakaryocytes (12-40 µm) with 1 nucleus; (3) micro-megakaryocytes (12-40 µm) with 2 nuclei; (4) micro-megakaryocytes (12-40 um) with multiple nuclei; (5) dys-morphic megakaryocytes (¡Ý40µm) with 1 nucleus; (6) dys-morphic megakaryocytes (¡Ý40 µm) with 2 nuclei; and (7) dys-morphic megakaryocytes (¡Ý40 µm) with multiple nuclei. To evaluate the prognostic impact of dys-megakaryopoiesis based on cell size we divided the seven subtypes into dys-megakaryopoiesis with and without micro-megakaryocytes. Samples were also divided based on numbers of nuclei: (1) mono-nucleated dys-morphic megakaryocytes; (2) bi-nucleated dys-morphic megakaryocytes; and (3) multinucleated dys-morphic megakaryocytes. The best discriminator cutoff point of each group was determined by the minimal P-value approach. The best discriminators were micro-megakaryocytes ¡Ý25%, dys-megakaryopoiesis except micro-megakaryocytes ¡Ý5%, mono-nucleated dys-megakaryopoiesis ¡Ý30% and bi-nucleated dys-megakaryopoiesis ¡Ý1%. In multi-nucleated megakaryopoiesis category, differences in survival at the optimal discriminator were not statistically significant (P=0.10). Results: Subjects in low- and high-risk cohorts were different with platelets (micro-megakaryocytes; P<0.001; dys-megakaryopoiesis except micro-megakaryocytes; P<0.001; mono-nucleated dys-megakaryopoiesis; P<0.001; bi-nucleated dys-megakaryopoiesis; P=0.028), bone marrow blasts (micro-megakaryocytes; P<0.001; dys-megakaryopoiesis except micro-megakaryocytes; P<0.001; mono-nucleated dys-megakaryopoiesis except micro-megakaryocytes; P<0.001; bi-nucleated dys-megakaryopoiesis; P<0.001), WHO 2008 subtypes (dys-megakaryopoiesis; P=0.001; dys-megakaryopoiesis except micro-megakaryocytes; P<0.001; mono-nucleated dys-megakaryopoiesis P<0.001; bi-nucleated dys-megakaryopoiesis; P=0.014) and IPSS-R risk cohorts (micro-megakaryocytes; P<0.001; dys-megakaryopoiesis except micro-megakaryocytes; P<0.001; mono-nucleated dys-megakaryopoiesis; P<0.001; bi-nucleated dys-megakaryopoiesis; P=0.001). There was no significant difference in age, gender, hemoglobin concentration and blood neutrophils levels at diagnosis between low- and high-risk cohorts. In addition, levels of micro-megakaryocytes and mono-nucleated megakaryocytes were significantly associated with IPSS-R cytogenetic category (P=0.002 and P=0.001). A significant association with IPSS-R cytogenetic category was not found for subjects with dys-megakaryopoiesis except micro-megakaryocytes and bi-nucleated megakaryopoiesis (P=0.187 and P=0.654).In multivariate analyses, micro-megakaryocytes ¡Ý25% and mono-nucleated dys-morphic megakaryocytes ¡Ý30% were independent adverse prognostic factors (hazard ratio [HR]=1.56 [95% confidence interval [CI], 1.10, 2.20]; P=0.012 and 1.49 [1.05, 2.10]; P =0.024). These effects were greater than those for other boundaries except micro-megakaryocytes ¡Ý5% and bi-nucleated dys-morphic megakaryocytes ¡Ý1% (P=0.288 and P =0.133). Conclusion: Our data suggest integration of micro-megakaryocytes and mono-nuclear dysmorphic megakaryocytes improves the predictive accuracy of the International Prognostic Scoring System-Revised (IPSS-R) scoring system. Disclosures No relevant conflicts of interest to declare.

Les mégacaryocytes sont les précurseurs cellulaires hautement spécialisés qui produisent des plaquettes via des extensions cytoplasmiques appelées proplaquettes. La formation des proplaquettes exige de profonds changements dans l’organisation du cytosquelette: microtubules et actine. Les formines sont une famille de protéines hautement conservées chez les eucaryotes composées de plusieurs domaines qui régulent le remodelage et la dynamique du cytosquelette d'actine et des microtubules. La plupart des formines sont des effecteurs protéiques des Rho-GTPase. DIAPH1, un membre de la famille des formines, est un homologue chez les mammifères du gène diaphanous de la drosophile qui fonctionne comme un effecteur de la petite GTPase Rho et régule le cytosquelette d'actomyosine ainsi que les microtubules. Il contient le domaine de liaison à Rho (Rho-binding domain) dans la partie amino-terminale et deux régions distinctes d’homologie aux formines, FH1 localisée au centre de la protéine et FH2 dans la partie carboxy-terminale. DIAPH1 co-régule le cytosquelette des microtubules et d'actine à travers respectivement ses régions de FH2 et FH1. DIAPH1 est donc un gène candidat idéal dans toutes les fonctions cellulaires qui exigent une coopération entre cytosquelettes d’actine et de microtubules.L'objectif de ce projet de thèse était d’étudier le rôle de DIAPH1 dans la mégacaryopoïèse. A la fin de la maturation des mégacaryocytes, la formation de proplquettes et la migration sont associées à des modifications importantes de la structure du cytosquelette. Nous avons émis l’hypothèse que grâce à la sa double fonction dans la polymérisation de l'actine et la stabilisation des microtubules, DIAPH1 pourrait jouer un rôle essentiel dans les temps terminaux de la différenciation mégacaryocytaire.Nos résultats ont montré qu’au cours de la différenciation mégacaryocytaire, l’expression de DIAPH1 augmente, alors que celles de DIAPH2 et DIAPH3 diminuent, ce qui suggère que DIAPH1 pourrait jouer un rôle plus important que DIAPH2 et DIAPH3 dans les stades tardifs de la différenciation mégacaryocytaire. Les études en immunomarquage montrent que DIAPH1 co-localise avec l’actine F, la tubuline et la myosine IIa en niveau de la membrane plasmique et des proplaquettes. Nous avons étudié la fonction de DIAPH1 par des stratégies d’invalidation (knockdown) et de surexpression d’une forme active de DIAPH1. Les résultats montrent que DIAPH1 est un effecteur important de Rho, pour réguler négativement la formation des proplaquettes en remodelant le cytosquelette d’actine et les microtubules. Le travail antérieur de notre équipe avait montré que Rho-ROCK régulait aussi négativement la formation des proplaquettes, en inhibant l’activation de la myosine IIa. En inhibant simultanément DIAPH1 et ROCK/myosine, nous avons montré que ces deux voies jouent un rôle additif dans la formation des proplaquettes.Ces résultats suggèrent que la coopération entre les voies DIAPH1 et ROCK/myosine est nécessaire pour la formation de structures cellulaire dépendant de l'actomyosine, telles les fibres de stress et l'anneau contractile en agissant à la fois sur le remodelage du cytosquelette et en assurant un équilibre entre l'actomyosine et microtubules
Megakaryocytes (MKs) are the highly specialized precursor cells that produce platelets via cytoplasm extensions called proplatelets. Proplatelet formation (PPF) requires profound changes in microtubule and actin organization. Formins are a family of highly conserved eukaryotic proteins with multidomains that govern dynamic remodeling of the actin and microtubule cytoskeletons. Most formins are Rho-GTPase effectors proteins. DIAPH1, a member of the formin family, is a mammalian homolog of Drosophila diaphanous gene that works as an effector of the small GTPase Rho and regulates the actomyosin cytoskeleton as well as microtubules. It contains the Rho-binding domain in the N-terminal and two distinct regions of formin homology, FH1 in the center and FH2 in the C-terminus. DIAPH coordinates microtubules and actin cytoskeleton through its FH2 and FH1 regions respectively, making DIAPH an ideal candidate in cell functions that depend closely on the cooperation between the actin and microtubule cytoskeletons.The objective of the project was to decipher the role of DIAPH1 in megakaryopoiesis. At the end of the MK maturation, PPF and MK migration are associated with profound changes in cytoskeleton organization. Due to its dual function in actin polymerization and microtubule stabilization, DIAPH1 was an obvious candidate to play an essential role in PPF and MK migration.Our results showed that DIAPH1 expression increased during MK differentiation, whereas DIAPH2 and DIAPH3 expression decreased, suggesting that DIAPH1 may play a more important role than DIAPH2 and DIAPH3 in the late stages of MK differentiation. Immunostaining showed that DIAPH1 co-localized with F-actin, tubulin and myosin IIa along the plasma membrane and proplatelet. Using a knockdown strategy with shRNA and expression of an active form of DIAPH1, we showed that DIAPH1 is an important effector of Rho that negatively regulates PPF by remodeling actin and microtubule cytoskeletons. A previous work of our team has shown that Rho-ROCK also negatively regulates in PPF by inhibiting myosin IIa activation. By the double inhibition of the DIAPH1 and the ROCK/Myosin pathway, we showed that DIAPH1 and ROCK played additive roles in the negative regulation of PPF. These observations suggest that the cooperation between DIAPH1 and ROCK is required for the formation of cell structures dependent on actomyosin, such as the stress fibers and the contractile ring. Collectively, these results strongly suggest that cooperation of DIAPH1/microtubules and ROCK/Myosin may regulate PPF by modifying the balance between actomyosin and microtubules

Megakaryocytes (Mks) are unique bone marrow cells, which produce platelets. Dysregulated Mk development can lead to abnormal platelet number and the production of functionally defective platelets, causing bleeding, thrombotic events, and leukaemia. Understanding the molecular mechanisms driving megakaryopoiesis may yield insights into the molecular genetics and cellular pathophysiology of a diversity of disorders. The primary aim of this thesis was to gain insight into the molecular events required for normal Mk development. As transcription factors and cytokines play a central role in driving Mk development, both of these processes were investigated. Fli-1 and GATA-1 are key transcription factors regulating Mk-gene expression, alone and co-operatively. To understand the mechanism of transcriptional synergy exerted by Fli-1 and GATA-1, in vitro assays were carried out investigating the interactions between Fli-1, GATA-1 and DNA that mediate synergy. A novel mechanism of synergy was identified, where Fli-1 DNA binding is not required, although an interaction between Fli-1 and GATA-1, and GATA-1 DNA binding is required. Importantly, the results demonstrate that Fli-1 DNA binding is not essential for promoting Mk-gene expression in primary murine bone marrow cells. Thrombopoietin (TPO) is the primary cytokine responsible for Mk and platelet development. Identifying novel TPO gene-targets may provide invaluable information to aid the understanding of the complex and unique processes required for Mk development. Using microarray technology, IFI16 was identified as a TPO-responsive gene that has not previously been studied in the Mk lineage. This work demonstrated that IFI16 is expressed in CD34+ HSC-derived Mks, and that the Jak/STAT pathway is essential for the activation of IFI16 by both TPO and IFN-??. Of biological significance, IFI16 was found to regulate both the proliferation and differentiation of primary Mks, suggesting that IFI16 may control the balance between these two essential processes. In conclusion, the data in this thesis presents a novel mechanism through which Fli-1 and GATA-1 regulate the synergistic activation of Mk genes. The identification and functional characterisation of a novel TPO-inducible gene, IFI16, involved in regulating the proliferation and differentiation of Mks is also described. These findings have implications for several congenital and malignant conditions affecting Mk and platelet development, and possibly a mechanism for IFN-induced thrombocytopaenia.

The immune adaptor ADAP possesses versatile roles in a variety of immune cells, including T cells, dendritic cells, macrophages, and platelets, etc. The most extensivelystudied role of ADAP is that it couples TCR activation to integrin activation and T-cell adhesion. However, the regulation of this adaptor during integrin activation and T-cell adhesion remains unclear. Meantime, the functions of ADAP linked to other immune cells are largely unknown. Work in this thesis have identified Ubc9, the sole SUMO E2 conjugase, as an essential regulator of ADAP in T-cell adhesion. We show that ADAP interacted directly with Ubc9 in vitro and in vivo, and the association was further strengthened in response to anti-CD3 stimulation. The Ubc9 binding domain on ADAP was mapped to a nuclear localisation sequence (aa 674-700) within ADAP. Knockdown of Ubc9 by shRNA or expression of the Ubc9-binding-deficient ADAP mutant significantly decreased TCRinduced integrin adhesion to ICAM-1 and fibronectin, as well as LFA-1 clustering, while having little effect on the TCR proximal signalling responses and TCR-induced IL-2 transcription. Furthermore, downregulation of Ubc9 impaired TCR-mediated Rac1 activation and attenuated the membrane targeting of Rap1 but not RIAM. Taken together, our data demonstrate for the first time that ADAP forms a functional interplay with Ubc9 and Ubc9 plays a selective role in integrin-mediated T-cell adhesion via modulation of Rap1 membrane recruitment and Rac1 activation. Another important finding of this thesis is the identification of a negative regulatory role for ADAP in the megakaryopoiesis. Here we show that in the bone marrow and spleen of ADAP-/- mice, a significant increase in the number of megakaryocytes were observed, and the ADAP-deficient megakaryocytes exhibited potentiated capacity in differentiation and development compared to the WT megakaryocytes. Mechanistically, ADAP directly interacted with STAT1, an indispensable modulator in megakaryopoiesis. Analysis on the activation of STAT1 showed that depletion of ADAP resulted in potentiated STAT1 phosphorylation and transcriptional activity, as well as upregulations of STAT1-regulatory genes. Collectively, these results suggest a novel role of ADAP in megakaryocytes, where ADAP attenuates megakaryopoiesis by direct interaction with STAT1 and negatively modulates the STAT1 activities. In summary, the work in this thesis have illustrated the diverse roles of ADAP in TCR-mediated integrin activation and megakaryopoiesis, and altogether contributed to our current knowledge of the many facets of ADAP in immunity.

Le processus qui aboutit à la formation de plaquettes est appelé mégacaryopoïèse. Les mégacaryocytes (MK) sont de grandes cellules de la moelle osseuse qui par fragmentation dans la circulation sanguine produisent des plaquettes. La régulation extrinsèque ou intrinsèque de ce processus a été largement étudiée. Cependant la régulation épigénétique reste mal connue bien que de nombreuses mutations dans des gènes de régulateurs épigénétiques soient retrouvées dans les hémopathies malignes de la lignée MK. En particulier des mutations du gène de la méthyltransférase EZH2, composant catalytique du Polycomb Repressive Complex 2 (PRC2) ont été détectées dans plusieurs types d’hémopathies. Ces mutations sont soit gain soit perte de fonction suggérant qu’EZH2 peut être à la fois un oncogène ou un gène suppresseur de tumeur. Dans les TE (Thrombocythémie Essentielle) et les MFP (Myélofibrose Primaire), deux néoplasmes myéloprolifératifs (NMPs), qui affectent principalement la lignée MK, des mutations d’EZH2 perte de fonction ont été retrouvées ainsi que dans les DS-AMKL (Down syndrome acute megakaryoblastic leukemia). Cela suggère qu’EZH2 joue un rôle important dans la mégacaryopoïèse normale. La caractérisation de cette fonction pourrait être utile pour mieux appréhender le rôle des mutations d’EZH2 dans les pathologies malignes mégacaryocytaires. Cette thèse peut être divisée en deux parties : 1) Caractérisation du rôle joué par EZH2 dans la mégacaryopoïèse normale et pathologique 2) Développement d‘un outil permettant d’étudier la coopération entre mutations dans les DS-AMKL.1) Lors des temps précoces de la différenciation in vitro des cellules CD34+ de sang de cordon vers la lignée mégacaryocytaire l’inhibition d’EZH2 entraîne l’acquisition plus rapide des marqueurs MK de surface (CD41 et CD42) pour un nombre de mitoses égal. Ceci suggère qu’EZH2 régule la spécification MK des progéniteurs hématopoïétiques. Plus tard dans la différenciation, l'inhibition constante d’EZH2 via des inhibiteurs ou des shRNA, arrête la prolifération et diminue le niveau de ploïdie des MKs en arrêtant la réplication de l’ADN. Ceci est du à la surexpression de plusieurs CDKi (Cyclin dependent kinase inhibiteurs), dont CDKN2D. L'analyse par Chip-Seq a montré que la transcription de CDKN2D est régulée par H3K27me3 au niveau de son promoteur et donc que CDKN2D est une nouvelle cible de PRC2. Dans les MKs les plus matures, l’inhibition d’EZH2 diminue la formation des proplaquettes, ceci est corrélé à des modifications d’expression de gènes régulant le cytosquelette d’actine. L’ensemble de ces résultats a été confirmé sur des MKs de patients porteurs de la mutation JAK2V617F.2) Par la technique CRISPR-Cas9, nous avons introduit dans des iPSC (induced pluripotent stem cells) disomiques et trisomiques pour le chromosome 21, la mutation GATA1s présente chez tous les patients avec une DS-AMKL. Nous avons montré que ces mutations modifiaient le cadre de lecture dans l’exon 2 et entrainaient l’expression de la forme courte de GATA1 (GATA1s). Nous sommes en train d'effectuer des études fonctionnelles ainsi que d’introduire d’autres mutations, y compris celles d’EZH2 pour modéliser la maladie.Au cours de cette thèse nous avons montré que l’inhibition d’EZH2 régule les temps initiaux de la mégacaryopoïèse en accélérant la spécification cellulaire au niveau des progéniteurs et ensuite la maturation terminale en inhibant profondément la polyploïdisation par surexpression de plusieurs CDKi dont CDKN2D et en inhibant la formation des plaquettes par un effet sur le cytosquelette d’actine. Ces résultats pourront être utiles pour mieux comprendre le rôle de la perte de fonction d’EZH2 dans les hémopathies malignes de la lignée mégacaryocytaire
The process that leads to platelet production is called megakaryopoiesis. Megakaryocytes (MK) are the large bone marrow cells that produce platelets by fragmentation in the blood flow. The extrinsic and intrinsic regulation of megakaryopoiesis has been largely studied. However, the epigenetic regulation remains poorly known although numerous mutations in genes of epigenetic regulators have been found in patients with MK hematological malignancies. The methyltransferase EZH2, the catalytic component of Polycomb Repressive Complex 2 (PRC2) is among the most studied epigenetic regulators. EZH2 is also mutated in many malignant hematological disorders where it can be an oncogene or a tumor suppressor gene. Particularly in ET (Essential Thrombocythemia) and PMF (Primary Myelofibrosis), two myeloproliferative neoplasms (MPNs) that affect mainly the MK lineage, loss of function EZH2 mutations have been found as well as in DS-AMKL (Down syndrome acute megakaryoblastic leukemia)Altogether these observations suggest that EZH2 controls normal megakaryopoiesis and characterization of this function could be helpful to understand the role of EZH2 in MK malignant diseases.This thesis can be divided in two parts:1) Characterization of the role of EZH2 in normal and pathological megakaryopoiesis 2) Establishment of a cellular tool to study the cooperation between the different mutations of DS-AMKL. RESULTS1) Using CD34+ cells isolated from cord blood, we showed that at early stages of differentiation, EZH2 inhibition accelerates the acquisition of MK surface markers (CD41a and CD42a) without increasing proliferation suggesting that EZH2 regulates the specification towards the MK lineage. Later in differentiation the constant inhibition of EZH2 via inhibitors or shRNAs, produced a proliferation arrest and a decrease in ploidy level that was related to an arrest in DNA replication due to an upregulation of several CDKi (Cyclin dependent kinase inhibitors), more particularly CDKN2D. Chip-Seq analysis demonstrated that CDKN2D is effectively regulated by H3K27me3 and is a new target of PRC2. This inhibition of ploidization by EZH2 inhibition was confirmed in MK from JAK2V617F patients. Furthermore in the more mature MKs (normal or JAK2V617F) we observed a defect in proplatelet formation, which was associated with an abnormal expression of genes regulating the actin filament. 2) By CRISPR-Cas 9, in iPSCs either disomic or chromosome 21 trisomic, we introduced, the GATA1s mutation present in all DS-AMKL patients. We confirmed at the gene and protein level that this genome editing has been correctly performed and that it induces as previously observed a blockage in erythroid differentiation. We are now carrying out the complete functional characterization together with the introduction of other mutations of DS-AMKL including EZH2.CONCLUSIONThis study describes EZH2 as a regulator of megakaryopoiesis via an initial control of cell specification and then of MK maturation. These results will be useful to better understand the role that EZH2 plays in diseases affecting the MK lineage such as MPNs and DS-AMKL

Investigations on platelet-derived growth factor (PDGF) and serotonin (5-HT), molecules stored in platelet granules, imply their potential effects in regulating megakaryopoiesis, which also intimates the existence of an autocrine and/or paracrine loop constructed by megakaryocytes/platelets and their granular constituents. In addition, numerous reports indicate that melatonin, a derivative from serotonin effectively enhances platelet counts in patients with thrombocytopenia. However, their exact roles on human megakaryocytes and the underlying mechanisms remain unknown. Present studies showed that PDGF, like thrombopoietin (TPO), significantly promoted platelet recovery and the formation of bone marrow colony-forming unit-megakaryocyte (CFU-MK) in an irradiated-mouse model. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow aspirate. In the M-07e apoptotic model, PDGF had a similar anti-apoptotic effect as TPO on megakaryocytes. Our findings demonstrated that PDGF activated the PI3-k/Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression. Our findings suggested that the PDGF-initiated radioprotective effect is likely to be mediated via PDGF receptors (PDGFRs) with subsequent activation of the PI3-k/Akt pathway. We also provide a possible explanation that blockade of PDGFR may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia. We explored how serotonin regulated megakaryopoiesis and proplatelet formation. Our results indicated that serotonin (5-HT) significantly promoted CFU-MK formation and reduced apoptosis on megakaryocytes through phosphorylation of Akt. These effects were attenuated by addition of ketanserin, a 5-HT2 receptor inhibitor. In addition, serotonin was able to stimulate the F-actin reorganization in megakaryocytes through activating the p-Erk1/2 expression. Bone marrow mesenchymal stromal cells (MSCs) are important in regulating megakaryopoiesis through stimulating the release of thrombopoietic growth factor, such as TPO. Our studies suggested that when activated by serotonin, bone marrow MSCs were induced to release significant amount of TPO. Furthermore, thousands of membrane-derived microparticles (MPs) arose from MSCs and the TPO RNA/proteins contained within MPs were also considerably increased under serotonin treatment. In summary, our findings demonstrated an important role serotonin played on megakaryopoiesis. This effect was likely mediated via 5HT2 receptors with subsequent activation of Akt and Erk 1/2 phosphorylation, which led to survival of megakaryocytes and proplatelet formation. Serotonin also stimulated TPO released from MSCs in both dissociative and MP-encapsulated form, which indirectly promoted megakaryopoiesis. The effects of melatonin on megakaryopoiesis were also determined in our studies. Our findings showed that melatonin enhanced proliferation and reduced doxorubicin-induced toxicity on MKs. We further demonstrated the mechanism for melatonin-mediated protection on MKs maybe via repair of G2/M phase cell cycle arrest and inhibition of cell apoptosis on MK cells. The effects of melatonin on megakaryopoiesis were also determined in our studies. Our findings showed that melatonin enhanced proliferation and reduced doxorubicin-induced toxicity on MKs. We further demonstrated the mechanism for melatonin-mediated protection on MKs maybe via repair of G2/M phase cell cycle arrest and inhibition of cell apoptosis on MK cells.
published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy

Megakaryocytes (MKs) are a rare population of haematopoietic cells, which produce platelets. Platelet production is a complex process that is tightly regulated at the transcriptional level by lineage specific transcription factors such as p45 NF-E2. Understanding how transcriptional regulators operate is imperative to advance our knowledge of disease pathophysiology and to propose novel treatment options. Therefore, the aims of this study were to: i) study the effects of p45 NF-E2 overexpression on various stages of megakaryopoiesis; (ii) elucidate the nuclear transport mechanisms of p45 NF-E2; and iii) determine the impact of a p45 NF-E2 modification called SUMOylation on thrombopoiesis. Exogenous p45 NF-E2 was overexpressed in haematopoietic cells in culture and various aspects of megakaryopoiesis were examined. Overexpression of p45 NF-E2 enhanced multiple stages of MK differentiation such as colony forming unit (CFU)-MK formation and terminal MK maturation. Most importantly, p45 NF-E2 overexpression resulted in significant increases in proplatelet and functional platelet production in vitro. This latter result was confirmed in vivo using lethally irradiated mice transplanted with cells that overexpressed p45 NF-E2. Unexpectedly, the enhancement of MK differentiation was at the expense of myeloid development and, for the first time, identified p45 NF-E2 as a negative regulator of myeloid differentiation. Secondly, we determined the nuclear localisation signal of p45-NF-E2 and the pathway responsible for nuclear import. We also investigated the importance of p45 NF-E2 nuclear import in thrombopoiesis. Finally, we showed that p45 NF-E2 is modified mainly by SUMO-2/3 in bone marrow cells and this process is involved in the transcriptional activation of MK-specific genes and platelet release. Taken together, these results suggest that enforced expression of p45 NF-E2 selectively enhances many aspects of MK differentiation including early and terminal MK maturation, proplatelet formation and platelet release. Equally important, this thesis also indicates that white blood cell differentiation may be inhibited by p45 overexpression, while molecular processes such as the nuclear import and SUMOylation of p45 NF-E2 are vital for thrombopoiesis. These observations will facilitate subsequent studies into the feasibility of manipulating p45 NF-E2 protein levels for the treatment of conditions such as thrombocytopaenia and other platelet disorders.

Thrombocytopenia is one of the most common hematologic presentations of active human cytomegalovirus (HCMV) infection, especially in recipients of allogeneic hematopoietic stem cell transplantations and newborns of congenital HCMV infection. However, mechanisms of HCMV-induced thrombocytopenia have not been well understood. The precursor of circulating platelets – megakaryocyte, is derived from hematopoietic stem/progenitor cell in bone marrow. We postulate that inhibition to megakaryocytic development is the major pathogenesis of HCMV-induced thrombocytopenia. Megakaryocytic cells as well as supportive microenvironment in bone marrow are major targets of HCMV infection. Presented study mainly focused on the impacts of HCMV to megakaryocytic cells and multipotent mesenchymal stromal cells (MSCs) - the precursor of bone marrow stromal cells. Based on a megakaryocytic cell model challenged by HCMV in vitro, inhibited megakaryocytic endomitosis, proliferation, and cellular expression were respectively demonstrated as decreased polyploidy population, decreased colony formation, and reduced c-Mpl (thrombopoietin receptor) expressing cells. Evoked apoptosis of megakaryocytic cells was also evidenced with increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation after HCMV infection. Involvement of mitochondrial-mediated intrinsic apoptosis was further shown as losing JC-1 fluorescent signal in infected megakaryocytic cells. These results suggest that inhibition induced by HCMV is exerted through multiple processes directly affecting the megakaryopoietic development. Functional failure of bone marrow microenvironment was demonstrated in bone marrow derived MSCs infected by HCMV in vitro. Suppressed cytokine production, impaired cellular migration, and hindered differentiation of HCMV-infected MSCs were respectively demonstrated by lowered level of stromal cell-derived factor 1 in culture medium, decreased number of cells passed through a porous membrane in a transwell culture, and reduced differentiated cells in either adipogenic or osteogenic induction cultures. Alongside with these changes, HCMV-induced programmed cell death further contributed to the supportive failure. Autophagic cell death in infected MSCs was demonstrated as massive accumulation of vacuoles with double membrane structure and LC-3b II molecules followed by viability loss. De novo apoptosis was also observed as another process of programmed cell death, shown as increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation of infected MSCs. Increased programmed cell death appeared to be associated with extensive HCMV replication in MSCs, which was featured with typical cytopathic morphology, expression of viral tegument protein pp65, and massive accumulation of various viral particles including mature virions. Sustained activation of extracellular signal-regulated kinases likely represented a signal transduction network connecting viral expression or replication with programmed cell death. In a “MSCs-dependent” megakaryopoiesis model, HCMV-infected MSCs failed to support survival and maintenance of megakaryocytic cells. Taken together, these results suggest that active HCMV expression or replication inhibits multiple cellular functions and induces multiple processes of programmed cell death of MSCs. Such inhibition compromises supportive functions of bone marrow microenvironment, and subsequently reduces platelet production in an indirect manner. In summary, HCMV suppresses cellular function and induced apoptosis on both megakaryocytic cells and their supportive cells, MSCs. Therefore, the inhibitory effects of HCMV on megakaryopoiesis are operated via both direct and indirect mechanisms.
published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy

Les néoplasmes myéloprolifératifs (NMPs) classiques BCR-ABL négatifs regroupent la Polyglobulie de Vaquez, la Thrombocytémie Essentielle et la Myélofibrose Primaire. Ce sont des pathologies malignes clonales entraînées par la signalisation constitutive de la voie JAK2/STAT en raison de mutations somatiques acquises qui affectent trois gènes, JAK2, CALR et MPL. Il s’agit des mutations “motrices” de la maladie responsable du syndrome myéloprolifératif et du phénotype. Cependant CALR n’est pas une molécule de signalisation mais une chaperonne du réticulum endoplasmique. En utilisant des lignées dépendantes de facteurs de croissance soit murines (Ba/F3) soit humaines (UT-7), des cellules primaires de patients et des modèles murins nous avons montré que les mutants CALRdel52 et CALRins5 avaient acquis de nouvelles propriétés qui en font des molécules de signalisation en induisant: - une indépendance aux facteurs de croissance uniquement lorsque MPL, le récepteur de la thrombopoïétine est exprimé ; - une phosphorylation constitutive de JAK2, des STAT1, 3 et 5 et une activation faible des voies PI3K/AKT et ERK1/2, suggérant une activation de MPL/JAK2 par les mutants CALR différente de celle induite par JAK2V617F. De manière intéressante un mutant de la CALR ayant une délétion entière de l’exon 9 n’est pas transformant, suggérant que l’activité oncogénique est liée à la présence de la nouvelle séquence C-terminale. L’activation de JAK2 uniquement par MPL en présence des mutants CALR pourrait expliquer le phénotype mégacaryocytaire/plaquettaire de ces NMPs. Cette activation de MPL au contraire de celle exercée par JAK2V617F a lieu non seulement à la membrane mais aussi dans le cytoplasme.Les modèles murins ont montré que les mutants CALR étaient responsables de la maladie et que celle-ci était dépendante de MPL, validant les résultats obtenus sur les lignées.Nous avons également montré que contrairement à JAK2V617F, les mutants de la CALR induisent chez l’homme une dominance clonale très tôt au niveau du compartiment des cellules souches. L’ensemble de ces résultats contribue à une meilleure compréhension du rôle des mutations CALR dans les NMPs. La démonstration que les molécules mutées sont présentes à la surface cellulaire ouvre la voie à des immunothérapies ciblant le nouveau peptide C-terminal
Classical BCR-ABL negative myeloproliferative neoplasms (MPNs) include three disorders: Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis. They are clonal malignant diseases driven by the constitutive JAK2/STAT signaling pathway due to acquired somatic mutations affecting three genes: JAK2, CALR and MPL. These are the "driver" mutations of the disease responsible of the myeloproliferation and of the disease phenotype. However, CALR is not a signaling molecule, but a chaperonne of the endoplasmic reticulum. Using murine (Ba/F3) and human (UT-7) cell lines dependent on growth factors and primary patient cells and mouse model, we have shown that the CALRdel52 and CALRins5 mutants have acquired new signaling properties and induce:- growth factor independence only when MPL, the thrombopoietin receptor, is expressed;- constitutive phosphorylation of JAK2, of STAT1, 3 and 5 and a low activation of the PI3K/AKT and ERK1/2 pathways, suggesting an activation of MPL/JAK2 by a different manner than JAK2V617F. Interestingly, a CALR mutant deleted for the entire exon 9 has not transformation properties suggesting that the oncogenic activity is related to the presence of the new C-terminal sequence. This JAK2 activation only by MPL in presence of CALR mutants could explain the megakaryocytic/platelet phenotype of these MPNs.The use of a mouse modeling using retroviral vectors and bone marrow transplantation has shown that CALRdel52 and ins5 were really the drivers of the disease and that in vivo the thrombocytosis was dependent of MPL validating the results obtained in vitro.In addition, we have shown that in human, CALR mutants induce a clonal dominance early in the stem cell compartment in ET. This is in sharp contrast with JAK2V617F in ET. Overall, these results contribute to a better comprehension of the role of CALR mutations in MPNs. Furthermore, the demonstration that the CALR mutants are expressed at the cell surface open the way to the development of new immunotherapy targetting the new C-terminus peptide

In this chapter, we discuss different topics always using the microRNA as the guiding thread of the review. MicroRNAs, member of small noncoding RNAs family, are an important element involved in gene expression. We cover different issues such as their importance in the differentiation and maturation of megakaryocytes (megakaryopoiesis), as well as the role in platelets formation (thrombopoiesis) focusing on the described relationship between miRNA and critical myeloid lineage transcription factors such as RUNX1, chemokines receptors as CRCX4, or central hormones in platelet homeostasis like TPO, as well as its receptor (MPL) and the TPO signal transduction pathway, that is JAK/STAT. In addition to platelet biogenesis, we review the microRNA participation in platelets physiology and function. This review also introduces the use of miRNAs as biomarkers of platelet function since the detection of pathogenic situations or response to therapy using these noncoding RNAs is getting increasing interest in disease management. Finally, this chapter describes the participation of platelets in cellular interplay, since extracellular vesicles have been demonstrated to have the ability to deliver microRNAs to others cells, modulating their function through intercellular communication, redefining the extracellular vesicles from the so-called “platelet dust” to become mediators of intercellular communication.

Thrombotic occlusion of microcirculation during DIC has already been studied in numerous organs, but little is known about analogous findings in the bone marrow. Therefore in rats under the influence of endotoxin the defibrination was examined for its relationship to changes inthe bone marrow and haemopoiesis. Bone marrow specimens were studied histologically by fibrin staining methods. Blood cells were measured automatically on the Coulter counter, fibrinogen by clotting assay. A fall in the thrombocyte and fibrinogen level was induced through a single injection of endotoxin with a maximum after 24 hours and 48 hours. In the same phase, after a short-term drop, a marked rise in the leukocyte count in the peripheral blood was observed. Following an initial increase the erythrocytes dropped and reached their lowest level after 48 hours. In the bone marrow 24 hours after endotoxin administration a large amount of fibrin deposits were observed in the small vessels. At the same time a clear reduction in all three haemopoietic cell lines was noticed. Between days five and ten the parameters of the peripheral blood normalized. Fibrin deposits in the bone marrow were no longer evident after three days. After 28 days an increase in the granulocytopoiesis and a continuing reduction in the megakaryopoiesis was observed.lt is concluded, due to the existence of fibrin deposits in the bone marrow in the beginning phase, that the observed haematologic alterations can not only be explained by the direct effect of endotoxin, but possibly also by the temporary microcircu-latory disturbances of the bone marrow during defibrination with its adverse effects on the haemopoiesis.